Photochromic xanthene fluorophores and their utility in live-cell imaging beyond the diffraction limit

ABSTRACT

The present invention is generally directed to novel fluorophores and their use in imaging methods. In one case, the present invention provides a compound according to the structure shown in FIG. 20A. In another case, the present invention provides a method of imaging one or more cellular structures within one or more cells using a compound of the structure shown in FIG. 20A.

FIELD OF THE INVENTION

The present invention is generally directed to novel fluorophores andtheir use in imaging methods.

BACKGROUND OF THE INVENTION

There have been reports of photochromic rhodamine dyes used in imagingmethods. For instance, Fölling et al. (“Fölling 1”) reports thesynthesis and examination of photochromic rhodamine derivatives.“Fluorescence Nanoscopy with Optical Sectioning by Two-Photon InducedMolecular Switching Using Continuous-Wave Lasers”, Angew. Chem. Int. Ed.2007, 46, 6266-6270. According to Folling 1, the rhodamine derivativeshave the following properties: “This readily controllablephotoswitchable compound has a high fluorescence quantum yield and highphotochemical stability under single-molecule conditions. The resultingdramatic increase in n yields an average localization precision ofapproximately 10 nm. In conjunction with an optimized asynchronous imageacquisition protocol, the large contrast between the two photochromicstates involved minimizes the diffuse background and allows us toabandon the total internal reflection (“TIRF”) recording schemes andmechanical object slicing that were mandatory in previous experiments.”Id. at 6266. A type of fluorescent compound discussed in this referenceis shown in FIG. 1.

Bossi et al. (“Bossi”) examines the photochromic switching of particularrhodamine amides, including their use in multicolorsingle-molecule-switching-based nanoscopy. “Multicolor Far-FieldFluorescence Nanoscopy Through Isolated Detection of Distinct MolecularSpecies”, Nano Lett. 2008, 8, 2463-2468. In the opinion of Bossi: “Bycombining the photoswitching and localization of individual fluorophoreswith spectroscopy on the single molecule level, we demonstratesimultaneous multicolor imaging with low crosstalk and down to 15 nmspatial resolution using only two detection color channels. Theapplicability of the method to biological specimens is demonstrated onmammalian cells. The combination of far-field fluorescence nanoscopywith the recording of a single switchable molecular species a at timeopens up a new class of functional imaging techniques.” Id. at Abstract.A type of fluorescent compound discussed in this reference is shown inFIG. 2.

Fölling et al. (“Fölling 2”) analyzes the photochromic reaction ofspecific rhodamine amides. “Fluorescence Nanoscopy with OpticalSectioning by Two-Photon Induced Molecular Switching UsingContinuous-Wave Lasers”, ChemPhysChem 2008, 9, 321-326. In the words ofFölling 2: “During the last decade far-field fluorescence microscopymethods have evolved that have resolution far below the wavelength oflight. To outperform the limiting role of diffraction, all thesemethods, in one way or another, switch the ability of a molecule to emitfluorescence. Here we present a novel rhodamine amide that can bephotoswitched from a nonfluorescent to a fluorescent state by absorptionof one or two photons from a continuous-wave laser beam. This brightmarker enables strict control of on/off switching and providessingle-molecule localization precision down to 15 nm in the focal plane.Two-photon induced non-linear photoswitching of this marker withcontinuous-wave illumination offers optical sectioning with simple laserequipment.” Id. at Abstract. A type of fluorescent compound discussed inthis reference is shown in FIG. 3.

Belov et al. (“Belov”) discusses distinct rhodamine spirolactamcompounds and their use in obtaining optical images. “RhodamineSpiroamides for Multicolor Single-Molecule Switching FluorescentNanoscopy”, Chem. Eur. J. 2009, 15, 10762-10776. As stated by Belov:“The design, synthesis, and evaluation of new rhodamine spiroamides aredescribed. These molecules have applications in optical nanoscopy basedon random switching of the fluorescent single molecules. The new markersmay be used in (co)localization studies of various objects and their(mutual) positions and shape can be determined with a precision of a fewtens of nanometers. Multicolor staining, good photoactivation, a largenumber of emitted photons, and selective chemical binding with amino orthiol groups were achieved due to the presence of various functionalgroups on the rhodamine spiroamides. Rigidized sulfonated xanthenefragment fused with six-membered rings, N,N′-bis(2,2,2-trifluoroethyl)groups, and a combination of additional double bonds and sulfonic acidgroups with simple aliphatic spiroamide residue provide multicolorproperties and improve performance of the rhodamine spiroamides inphotoactivation and bioconjugation reactions.” Id at Abstract. A type offluorescent compound discussed in this reference is shown in FIG. 4.

Lee et al. (“Lee”) discusses the use of certain rhodamine spirolactamderivatives. “Small-Molecule Labeling of Live Cell Surfaces forThree-Dimensional Super-Resolution Microscopy”, J. Am. Chem. Soc.2014,136, 14003-14006. Lee states: “Precise imaging of the cell surfaceof fluorescently labeled bacteria requires super-resolution methodsbecause the size-scale of these cells is on the order of the diffractionlimit. In this work, we present a photocontrollable small-moleculerhodamine spirolactam emitter suitable for non-toxic and specificlabeling of the outer surface of cells for three-dimensional (3D)super-resolution (SR) imaging. Conventional rhodamine spirolactamsphotoswitch to the emitting form with UV light; however, thesewavelengths can damage cells. We extended photo-switching to visiblewavelengths >400 nm by iterative synthesis and spectroscopiccharacterization to optimize the substitution on the spirolactam.” Id.at Abstract. A type of fluorescent compound discussed in this referenceis shown in FIG. 5.

Wan et. al. (“Wan”) reports a rhodamine B-based fluorescent probe andits application for the detection of trivalent ions. “Cascade Off-On-OffFluorescent Probe: Duel Detection of Trivalent Ions and Phosphate Ions”,RSC Adv. 2014, 4, 29479-29484. According to Wan: “A new rhodamineB-based fluorescent probe was developed for the selective cascadesignaling of trivalent cations (Fe³⁺, Al³⁺, Cr³⁺) and phosphate anion(PO₄ ³⁻). Non-fluorescent rhodamine derivatives can selectively detecttrivalent cations over some other metal ions in CH₃CN-Tris buffer (1/1,v/v, pH 7.0) solutions, leading to prominent fluorescence OFF-ONswitching. The obtained probecation complex can subsequently serve as asensitive and selective chemosensor for PO₄ ³⁻, exhibiting completesignal quenching (fluorescence ON-OFF switching).” Id. at Abstract. Atype of fluorescent compound discussed in this reference is shown inFIG. 6.

Despite reports of photochromic rhodamine dyes used in imaging methods,there is still a need in the art for novel fluorophores and their use inimaging methods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a type of fluorescent compound discussed in Fölling et al.“Fluorescence Nanoscopy with Optical Sectioning by Two-Photon InducedMolecular Switching Using Continuous-Wave Lasers”, Angew. Chem. Int. Ed.2007, 46, 6266-6270.

FIG. 2 shows a type of fluorescent compound discussed in Bossi et al.“Multicolor Far-Field Fluorescence Nanoscopy Through Isolated Detectionof Distinct Molecular Species”, Nano Lett. 2008, 8, 2463-2468.

FIG. 3 shows a type of fluorescent compound discussed in Fölling et al.“Fluorescence Nanoscopy with Optical Sectioning by Two-Photon InducedMolecular Switching Using Continuous-Wave Lasers”, ChemPhysChem 2008, 9,321-326.

FIG. 4 shows a type of fluorescent compound discussed in Belov et al.“Rhodamine Spiroamides for Multicolor Single-Molecule SwitchingFluorescent Nanoscopy”, Chem. Eur. J. 2009, 15, 10762-10776.

FIG. 5 shows a type of fluorescent compound discussed in Lee et al.“Small-Molecule Labeling of Live Cell Surfaces for Three-DimensionalSuper-Resolution Microscopy”, J Am. Chem. Soc. 2014,136, 14003-14006.

FIG. 6 shows a type of fluorescent compound discussed in Wan et. al.“Cascade Off-On-Off Fluorescent Probe: Duel Detection of Trivalent Ionsand Phosphate Ions”, RSC Adv. 2014, 4, 29479-29484.

FIG. 7 shows a general structure for compounds according to the presentinvention.

FIG. 8 shows nonlimiting examples of Acceptor moieties included incompounds according to the present invention.

FIG. 9 shows four different moieties that can, individually, be elementsY₁ and/or Y₂ in the structure of FIG. 7.

FIGS. 10-13 show examples of structures of compounds according to thepresent invention.

FIG. 14 shows a super-resolution image of a nucleus using PALM and acompound according to the present invention.

FIGS. 15-17 show schemes for the synthesis of compounds according to thepresent invention.

FIGS. 18-20 show general structures for compounds according to thepresent invention.

FIGS. 21-27 show further schemes for the synthesis of compoundsaccording to the present invention.

FIGS. 28-33 show further super-resolution images using compoundsaccording to the present invention.

SUMMARY OF THE INVENTION

In one case, the present invention provides a compound according to thestructure shown in FIG. 20A. The elements of the shown structure aredefined as follows: X is O, N-alkyl, S, Si(alkyl)₂ or C(alkyl)₂; Y₁ isO, OH, NH₂, N(alkyl)₂ or one of the moieties shown in FIG. 9 (i.e., a,b, c, or d); Y₂ is O, OH, NH₂, N(alkyl)₂ or one of the moieties shown inFIG. 9 (i.e., a, b, c, or d); R₁, which can be a substitution at eitherthe 5′ position, the 6′ position or both, is hydrogen, C(O)NH-Handle,C(O)-linker-Handle, C(O)NH-Acceptor, C(O)-linker-Acceptor,C(O)NH-linker-CH2-X where X is a leaving group,C(O)NH—(CH₂CH₂)_(n)C(O)N(H)-Handle where “n” is an integer ranging from1 to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” is an integer rangingfrom 1 to 100, C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH2)₆—Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆—Cl; R₂ ishydrogen, C(O)NH-Handle, C(O)-linker-Handle, C(O)NH-Acceptor,C(O)-linker-Acceptor, C(O)NH-linker-CH2-X where X is a leaving group,C(O)NH—(CH₂CH₂)_(n)C(O)N(H)-Handle where “n” is an integer ranging from1 to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” is an integer rangingfrom 1 to 100, C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH2)₆—Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆—Cl; R₃, R₄,R₅ and R₆ are independently hydrogen, alkyl, —SO₃H, halogen, or R₃ andY₁ can form a ring, or R₄ and Y₁ can form a ring, or R₅ and Y₂ can forma ring, or R₆ and Y₂ can form a ring; A₁ is hydrogen, alkyl, alkenyl,alkynyl, heteroalkyl, heteroalkenyl, cycloalkyl, cycloalkenyl,heterocycloalkyl, heterocycloalkenyl, aromatic, heteroaromatic,substituted alkyl, substituted alkenyl, substituted alkynyl, substitutedheteroalkyl, substituted heteroalkenyl, heterocycloalkyl,heterocycloalkenyl, substituted aromatic group, or substitutedheteroaromatic group; A₂ is hydrogen, alkyl, alkenyl, alkynyl,heteroalkyl, heteroalkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl,heterocycloalkenyl, aromatic, heteroaromatic, substituted alkyl,substituted alkenyl, substituted alkynyl, substituted heteroalkyl,substituted heteroalkenyl, heterocycloalkyl, heterocycloalkenyl,substituted aromatic group, or substituted heteroaromatic group.

In another case, the present invention provides a method of imaging oneor more cellular structures within one or more cells. The methodincludes the steps of:

a) labeling one or more cells with a compound according to the structureshown in FIG. 20A, where the elements of the shown structure are definedas follows: X is O, N-alkyl, S, Si(alkyl)₂ or C(alkyl)₂; Y₁ is O, OH,NH₂, N(alkyl)₂ or one of the moieties shown in FIG. 9 (i.e., a, b, c, ord); Y₂ is O, OH, NH₂, N(alkyl)₂ or one of the moieties shown in FIG. 9(i.e., a, b, c, or d); R₁, which can be a substitution at either the 5′position, the 6′ position or both, is hydrogen, C(O)NH-Handle,C(O)-linker-Handle, C(O)NH-Acceptor, C(O)-linker-Acceptor,C(O)NH-linker-CH2-X where X is a leaving group,C(O)NH—(CH₂CH₂)_(n)C(O)N(H)-Handle where “n” is an integer ranging from1 to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” is an integer rangingfrom 1 to 100, C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂-(CH2)₆—Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆—Cl; R₂ ishydrogen, C(O)NH-Handle, C(O)-linker-Handle, C(O)NH-Acceptor,C(O)-linker-Acceptor, C(O)NH-linker-CH2-X where X is a leaving group,C(O)NH—(CH₂CH₂)_(n)C(O)N(H)-Handle where “n” is an integer ranging from1 to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” is an integer rangingfrom 1 to 100, C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂-(CH2)₆-Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆—Cl; R₃, R₄,R₅ and R₆ are independently hydrogen, alkyl, —SO₃H, halogen, or R₃ andY₁ can form a ring, or R₄ and Y₁ can form a ring, or Rand Y₂ can form aring, or R₆ and Y₂ can form a ring; A₁ hydrogen, alkyl, alkenyl,alkynyl, heteroalkyl, heteroalkenyl, cycloalkyl, cycloalkenyl,heterocycloalkyl, heterocycloalkenyl, aromatic, heteroaromatic,substituted alkyl, substituted alkenyl, substituted alkynyl, substitutedheteroalkyl, substituted heteroalkenyl, heterocycloalkyl,heterocycloalkenyl, substituted aromatic group, or substitutedheteroaromatic group; A₂ is hydrogen, alkyl, alkenyl, alkynyl,heteroalkyl, heteroalkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl,heterocycloalkenyl, aromatic, heteroaromatic, substituted alkyl,substituted alkenyl, substituted alkynyl, substituted heteroalkyl,substituted heteroalkenyl, heterocycloalkyl, heterocycloalkenyl,substituted aromatic group, or substituted heteroaromatic group.

to provide one or more labeled cells

b) directing at least one beam of light to the one or more labeledcells, such that a detectable signal is produced from the one or morelabeled cells;

c) recording the detectable signal, thereby imaging one or morestructures within the one or more cells.

DETAILED DESCRIPTION OF THE INVENTION

“Acceptor” refers to a chemical entity that has a high affinity forelectrons. It is typically a chemical entity that has at least oneelectron withdrawing group attached to another portion of a moleculethrough a point of unsaturation—e.g., olefin.

“Alkane” refers to an acyclic saturated hydrocarbon and having thegeneral formula C_(n)H_(2n+2). Examples of lower alkanes (C₁-C₅)include: methane; ethane; propane; butane; and pentane. Other,nonlimiting examples of alkanes are: hexane; heptane; octane; nonane;and decane.

“Alkyl” refers to an alkane missing one hydrogen and having the generalformula C_(n)H_(2n+1). Examples of lower alkyls (C1-C5) include: methyl;ethyl; propyl; butyl; and pentyl. Other, nonlimiting examples of alkylsare: hexyl; heptyl; octyl; nonyl; and decyl.

“Alkene” refers to an unsaturated hydrocarbon that contains at least onecarbon-carbon double bond. Examples of lower alkenes (C2-C5) include:ethene; propene; butene; and pentene. Other, nonlimiting examples ofalkenes include: hexene; heptene; octene; nonene; and decene.

“Alkenyl” refers to an alkene missing one hydrogen. Examples of loweralkenyls (C2-C5) include: ethenyl; propenyl; butenyl; and pentenyl.Other, nonlimiting examples of alkenyls include: hexenyl; heptenyl;octenyl; nonenyl; and decenyl.

“Alkyne” refers to an unsaturated hydrocarbon that contains at least onecarbon-carbon triple bond. Examples of lower alkynes (C2-C5) include:ethyne; propyne; butyne; and pentyne. Other, nonlimiting examples ofalkynes include: hexyne; heptyne; octyne; nonyne; and decyne.

“Alkynyl” refers to an alkyne missing one hydrogen. Examples of loweralkynyls (C2-C5) include: ethynyl; propynyl; butynyl; and pentynyl.Other, nonlimiting examples of alkynyls include: hexynyl; heptynyl;octynyl; nonynyl; and decynyl.

“Aromatic group” refers to a cyclic or multi-cyclic, planar moleculewith a ring of resonance bonds that exhibit more stability than othergeometric or connective arrangements with the same set of atoms.Nonlimiting examples of aromatic groups include: phenyl; naphthyl;anthracenyl; and phenanthrenyl.

“Cycloalkane” refers to an alkane arranged in a ring structure. Thegeneral formula for a cycloalkane is C_(n)H_(2(n+1−r)). Nonlimitingexamples of cycloalkanes include: cyclopropane; cyclobutane;cyclopentane; cyclohexane; cycloheptane; cyclooctane; cyclononane; andcyclodecane.

“Cycloalkene” refers to an alkene arranged in a ring structure.Nonlimiting examples of cycloalkenes include: cyclopropene; cyclobutene;cyclopentene; cyclohexene; cycloheptene; cyclooctene; cyclononene; andcyclodecene.

“Cycloalkenyl” refers to a cycloalkane missing one hydrogen atom.Nonlimiting examples of cycloalkenyls include: cyclopropenyl;cyclobutenyl; cyclopentenyl; cyclohexenyl; cycloheptenyl; cyclooctenyl;cyclononenyl; and cyclodecenyl.

“Cycloalkyl” refers to a cycloalkane missing one hydrogen and having thegeneral formula C_(n)H_(2n−1). Nonlimiting examples of cycloalkylsinclude: cyclopropyl; cyclobutyl; cyclopentyl; cyclohexyl; cycloheptyl;cyclooctyl; cyclononyl; and cyclodecyl.

“Electron withdrawing group” (i.e., “EWG”) refers to an individual atomor functional group that withdraws electron density from a conjugatedsystem. Nonlimiting examples of electron withdrawing groups include:—CN; —C(O)H; —C(O)-alkyl; —CO₂H; —CO₂-alkyl; —NO₂; —S(O)-alkyl;—S(O)₂-alkyl; and —SO₃H.

“HaloTag” refers to a protein tag including a modified haloalkanedehalogenase designed to covalently bind to synthetic ligands. Thesynthetic ligands comprise a chloroalkane linker attached to a varietyof molecules. Nonlimiting examples of such molecules include: biotin;fluorescent dyes (e.g., Coumarin, Oregon Green, Alexa Fluor 488, diAcFAMand TMR); affinity handles; and solid surfaces. See, for example, Los etal., “A Novel Protein Labeling Technology for Cell Imaging and ProteinAnalysis”, ACS Chem. Biol. 2008, 3, 373-382, which isincorporated-by-reference into this document for all purposes.

“Handle” refers to a biomolecule tag. Nonlimiting examples of handlesinclude: a HaloTag; a SnapTag; a TMPTag; an NHS ester; and aβ-lactamase.

“Heteroalkane” refers to an alkane, where one or more of the carbonatoms in the alkane is replaced by a heteroatom (e.g., O, S, N-alkyl).Nonlimiting examples of heteroalkanes include: CH₃OCH₃; CH₃SCH₃;CH₃N(CH₃)CH₃; CH₃OCH₂CH₃; CH₃SCH₂CH₃; CH₃N(CH₃)—CH₂CH₃; CH₃CH₂OCH₂CH₃;CH₃CH₂SCH₂CH₃; CH₃CH₂N(CH₃)CH₂CH₃; CH₃CH₂O—(CH₂CH₂O)_(n)CH₃, where n isan integer ranging from 2 to 100; and CH₃CH₂O—(CH₂CH₂O)_(n)—CH₂CH₃,where n is an integer ranging from 2 to 100.

“Heteroalkyl” refers to a heteroalkane missing one hydrogen. Nonlimitingexamples of heteroalkyls include: CH₂OCH₃; CH₂SCH₃; CH₂N(CH₃)₂;CH₂OCH₂CH₃; CH₂SCH₂CH₃; CH₂N(CH₃)—CH₂CH₃; CH₂CH₂OCH₂CH₃; CH₂CH₂SCH₂CH₃;CH₂CH₂N(CH₃)CH₂CH₃; CH₂CH₂O(CH₂CH₂O),CH₃, where n is an integer rangingfrom 2 to 100; and CH₂CH₂O—(CH₂CH₂O)_(n)CH₂CH₃, where n is an integerranging from 2 to 100.

“Heteroalkene” refers to an alkene, where one or more carbon atoms inthe alkene is replaced by a heteroatom (e.g., O, S, N-alkyl).Nonlimiting examples of heteroalkenes include: CH₂CHCH₂OCH₃;CH₂CHCH₂SCH₃; CH₂CHCH₂N(CH₃)₂; CH₂CHCH₂CH₂OCH₃; CH₂CH—CH₂CH₂SCH₃; andCH₂CHCH₂CH₂N(CH₃)₂.

“Heteroalkenyl” refers to a heteroalkene missing one hydrogen.Nonlimiting examples of heteroalkenes include: CHCHCH₂OCH₃; CHCHCH₂SCH₃;CHCHCH₂N(CH₃)₂; CHCHCH₂CH₂OCH₃; CHCH—CH₂CH₂SCH₃; and CHCHCH₂CH₂N(CH₃)₂.

“Heterocycloalkane” refers to a cycloalkane where one or more carbonatoms in the cycloalkane is replaced by a heteroatom (e.g., O, S,N-alkyl). Nonlimiting examples of heterocycloalkanes include: (CH₂)₄O;(CH₂)₄S; (CH₂)₄N—CH₃; (CH₂)₅O; (CH₂)₅S; and (CH₂)₅N—CH₃.

“Heterocycloalkyl” refers to a heterocycloalkane missing one hydrogen.Nonlimiting examples of heterocycloalkyls include: CH(CH₂)₃O; CH(CH₂)₃S;CH(CH₂)₃NCH₃; CH(CH₂CH₂)₂O; CH(CH₂CH₂)₂S; and CH(CH₂CH₂)NCH₃.

“Heteroaromatic group” refers to an aromatic group where one or more ofthe carbon atoms has been replaced by a heteroatom (e.g., N). refers toa cyclic or multi-cyclic, planar molecule with a ring of resonance bondsthat exhibit more stability than other geometric or connectivearrangements with the same set of atoms, wherein the cyclic ormulti-cyclic, planar molecule contains a heteroatom (e.g., O, S, N).Nonlimiting examples of heteroaromatic groups include: furanyl;thiophenyl; pyrrolyl; and pyridyl.

“β-Lactamase Tag” refers to the combination of a mutant β-lactamase tagwith a fluorophore-derivatized probe. In use, the tag is covalentlybound to a target protein. See, for example, Watanabe et al.,“Multicolor Protein Labeling in Living Cells Using Mutantβ-Lactamase-Tag Technology”, Bioconjug Chem 2010, 21, 2320-2326.

“Leaving group” refers to a chemical moiety that is capable of beingdisplaced by a nucleophilic compound, typically through an S_(N)2reaction. Nonlimiting examples of leaving groups include: Cl, Br, I,OAc, methyl sulfate ion, methanesulfonate ion,trifluoromethane-sulfonate ion, and 4-methylbenzenesulfonate ion.

“Linker” refers to a chemical group that connects two parts of amolecule, typically through covalent bonds. A generic example of alinker is: A-linker-B, where “A” and “B” are parts of a molecule, andthe bifunctional linker (e.g., linker has functional groups at bothtcrmini that are capable of covalently binding to moieties on themolecular parts it connects) is covalently bonded to part “A” on oneterminus and part “B” on the other. Nonlimiting examples of linkersinclude: C(O)-alkyl-NH; C(O)-alkyl-N(alkyl); C(O)-alkyl-O; C(O)-alkyl-S;NH-alkyl-NH; N(alkyl)-alkyl-NH; N(alkyl)-alkyl-N(alkyl); NH-alkyl-O;N(alkyl)-alkyl-O; NH-alkyl-S; N(alkyl)-alkyl-S;O-(CH₂CH₂O)_(n)—CH₂CH₂OH, where “n” is an integer ranging from 1 to 100;O—(CH₂CH₂O)_(n)—CH₂CH₂O-alkyl, where “n” is an integer ranging from 1 to100; NH—(CH₂CH₂O)_(n)—CH₂CH₂OH, where “n” is an integer ranging from 1to 100; and NH—(CH₂CH₂O)_(n)—CH₂CH₂O—alkyl, where “n” is an integerranging from 1 to 100.

“NHS Ester” refers to an N-hydroxysuccinimide ester. NHS esters arereactive groups formed by carbodiimide-activation of carboxylatemolecules. NHS ester-activated crosslinkers and labeling compounds reactwith primary amines in physiologic to slightly alkaline conditions (pH7.2 to 9) to yield stable amide bonds. The reaction releasesN-hydroxysuccinimide.

“Photoactivated localization microscopy” (PALM) refers to a form ofsuper-resolution microscopy. PALM imaging pin-points individualmolecules in a sample and subsequently reconstructs an extremely highresolution image from hundreds of frames. This type of microscopy iscapable of nanometer scale resolution through the use ofphoto-switchable fluorophores. In operation, a low-power activatinglaser beam randomly converts the fluorophore to an active state; theactive state molecules are imaged by a high-power illuminating laserbeam to immediately convert them back to an inactive state. Theactive/inactive conversion is repeated over thousands of frames suchthat all the fluorophores have been imaged. See, for example, Betzig etal. “Imaging Intracellular Fluorescent Proteins at NanometerResolution”, Science 2006, 313, 1642-1645, which isincorporated-by-reference into this document for all purposes.

“SnapTag” refers to a self-labeling protein tag commercially availablein various expression vectors. It is a 182 residue polypeptide that iscapable of being fused to a targeted protein and covalently bound to aligand (e.g., fluorescent dye). See Crivat et al., “Imaging ProteinsInside Cells with Fluorescent Tags”, Trends in Biotechnology 2012, 30,8-16, which is incorporated-by-reference into this document for allpurposes.

“Substituted alkyl” refers to an alkyl where one or more hydrogen atomshave been replaced with a different substituent. Nonlimiting examples ofsuch substituents include: alkyl; alkenyl; alkynyl; cycloalkyl;cycloalkenyl; heterocycloalkyl; heterocycloalkenyl; aromatic group;heteroaromatic group; OH; O-alkyl; NH₂; NH-alkyl; SH; CN; NO₂; CF₃;C(O)H; C(O)-alkyl; CO₂H; CO₂-alkyl; OC(O)CH₃.

“Substituted alkenyl” refers to an alkenyl where one or more hydrogenatoms have been replaced with a different substituent. Nonlimitingexamples of such substituents include: alkyl; alkenyl; alkynyl;cycloalkyl; cycloalkenyl; heterocycloalkyl; heterocycloalkenyl; aromaticgroup; heteroaromatic group; OH; O-alkyl; NH₂; NH-alkyl; SH; CN; NO₂;CF₃; C(O)H; C(O)-alkyl; CO₂H; CO₂-alkyl; OC(O)CH₃.

“Substituted aromatic group” refers to an aromatic group where one ormore hydrogen atoms have been replaced with a different substituent.Nonlimiting examples of such substituents include: alkyl; alkenyl;alkynyl; cycloalkyl; cycloalkenyl; heterocycloalkyl; heterocycloalkenyl;aromatic group; heteroaromatic group; OH; O-alkyl; NH₂; NH-alkyl; SH;CN; NO₂; CF₃; C(O)H; C(O)-alkyl; CO₂H; CO₂-alkyl; OC(O)CH₃.

“Substituted cycloalkenyl” refers to a cycloalkenyl where one or morehydrogen atoms have been replaced with a different substituent.Nonlimiting examples of such substituents include: alkyl; alkenyl;alkynyl; cycloalkyl; cycloalkenyl; heterocycloalkyl; heterocycloalkenyl;aromatic group; heteroaromatic group; OH; O-alkyl; NH₂; NH-alkyl; SH;CN; NO₂; CF₃; C(O)H; C(O)-alkyl; CO₂H; CO₂-alkyl; OC(O)CH₃.

“Substituted cycloalkyl” refers to a cycloalkyl where one or morehydrogen atoms have been replaced with a different substituent.Nonlimiting examples of such substituents include: alkyl; alkenyl;alkynyl; cycloalkyl; cycloalkenyl; heterocycloalkyl; heterocycloalkenyl;aromatic group; heteroaromatic group; OH; O-alkyl; NH₂; NH-alkyl; SH;CN; NO₂; CF₃; C(O)H; C(O)-alkyl; CO₂H; CO₂-alkyl; OC(O)CH₃.

“Substituted heteroalkyl” refers to a heteroalkyl where one or morehydrogen atoms have been replaced with a different substitucnt.Nonlimiting examples of such substituents include: alkyl; alkenyl;alkynyl; cycloalkyl; cycloalkenyl; heterocycloalkyl; heterocycloalkenyl;aromatic group; heteroaromatic group; OH; O-alkyl; NH₂; NH-alkyl; SH;CN; NO₂; CF₃; C(O)H; C(O)-alkyl; CO₂H; CO₂-alkyl; OC(O)CH₃.

“Substituted heteroalkenyl” refers to a heteroalkenyl where one or morehydrogen atoms have been replaced with a different substituent.Nonlimiting examples of such substituents include: alkyl; alkenyl;alkynyl; cycloalkyl; cycloalkenyl; heterocycloalkyl; heterocycloalkenyl;aromatic group; heteroaromatic group; OH; O-alkyl; NH₂; NH-alkyl; SH;CN; NO₂; CF₃; C(O)H; C(O)-alkyl; CO₂H; CO₂-alkyl; OC(O)CH₃.

“Substituted heteroaromatic group” refers to a heteroaromatic where oneor more hydrogen atoms have been replaced with a different substituent.Nonlimiting examples of such substituents include: alkyl; alkenyl;alkynyl; cycloalkyl; cycloalkenyl; heterocycloalkyl; heterocycloalkenyl;aromatic group; heteroaromatic group; OH; O-alkyl; NH₂; NH-alkyl; SH;CN; NO₂; CF₃; C(O)H; C(O)-alkyl; CO₂H; CO₂-alkyl; OC(O)CH₃.

“Substituted heterocycloalkenyl” refers to a heterocycloalkenyl whereone or more hydrogen atoms have been replaced with a differentsubstituent. Nonlimiting examples of such substituents include: alkyl;alkenyl; alkynyl; cycloalkyl; cycloalkenyl; heterocycloalkyl;heterocycloalkenyl; aromatic group; heteroaromatic group; OH; O-alkyl;NH₂; NH-alkyl; SH; CN; NO₂; CF₃; C(O)H; C(O)-alkyl; CO₂H; CO₂-alkyl;OC(O)CH₃.

“Substituted heterocycloalkyl” refers to a heterocycloalkyl where one ormore hydrogen atoms have been replaced with a different substituent.Nonlimiting examples of such substituents include: alkyl; alkenyl;alkynyl; cycloalkyl; cycloalkenyl; heterocycloalkyl; heterocycloalkenyl;aromatic group; heteroaromatic group; OH; O-alkyl; NH₂; NH-alkyl; SH;CN; NO₂; CF₃; C(O)H; C(O)-alkyl; CO₂H; CO₂-alkyl; OC(O)CH₃.

“Super-resolution microscopy” refers a form of light microscopy thatallows images to be taken with a higher resolution than the diffractionlimit. See, for example, Neice et al. “Methods and Limitations ofSubwavelength Imaging”, Advances in Imaging and Electron Physics 2010,163, 117-140, which is incorporated-by-reference into this document forall purposes.

“TMPTag” refers to use of a trimethorprim derivative as a tag to labelbiomolecules (e.g., proteins). Use of TMPTags is typically carried outas follows: A target protein is tagged with an E. coli dihydrofolatereductase cysteine mutant and covalently bound to a cell-permeableacrylamide-trimethoprim-fluorophore. See, for example Miller et al. “InVivo Protein Labeling with Trimethoprim Conjugates: a Flexible ChemicalTag”, Nat Methods 2005, 2, 255-257 and Wang et al. “The CovalentTrimethoprim Chemical Tag Facilitates Single Molecule Imaging withOrganic Fluorophores”, Biophysical Journal 2014, 106, 272-278, both ofwhich are incorporated-by-reference into this document for all purposes.

FIG. 7 shows a general structure for compounds according to the presentinvention, where the elements X, Y₁, Y₂, R₁, R₂, A₁, A₂ and Z aredefined as follows:

X is O, N-alkyl, S, Si(alkyl)₂ or C(alkyl)₂;

Y₁ is O, N(alkyl)₂ or one of the moieties shown in FIG. 9 (i.e., a, b,c, or d);

Y₂ is O, N(alkyl)₂ or one of the moieties shown in FIG. 9 (i.e., a, b,c, or d);

R₁ is hydrogen, C(O)NH-Handle, C(O)-linker-Handle, C(O)NH-Acceptor,C(O)-linker-Acceptor, C(O)NH-linker-CH2-X where X is a leaving group,C(O)NH—(CH₂CH₂)_(n)C(O)N(H)-Handle where “n” is an integer ranging from1 to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” is an integer rangingfrom 1 to 100, C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH2)₆—Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆—Cl;

R₂ is hydrogen, C(O)NH-Handle, C(O)-linker-Handle, C(O)NH-Acceptor,C(O)-linker-Acceptor, C(O)NH-linker-CH2-X where X is a leaving group,C(O)NH—(CH₂CH₂)_(n)C(O)N(H)-Handle where “n” is an integer ranging from1 to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” is an integer rangingfrom 1 to 100, C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂-(CH2)₆-Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆—Cl;

A₁ is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl,aromatic, heteroaromatic, substituted alkyl, substituted alkenyl,substituted alkynyl, substituted heteroalkyl, substituted heteroalkenyl,heterocycloalkyl, heterocycloalkenyl, substituted aromatic group, orsubstituted heteroaromatic group;

A₂ is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl,aromatic, heteroaromatic, substituted alkyl, substituted alkenyl,substituted alkynyl, substituted heteroalkyl, substituted heteroalkenyl,heterocycloalkyl, heterocycloalkenyl, substituted aromatic group, orsubstituted heteroaromatic group;

Z is O, S, Se, Te or an Acceptor.

FIG. 8 shows nonlimiting examples of Acceptor moieties included incompounds according to the present invention.

Nonlimiting examples of compounds according to the present invention are(in reference to FIG. 7):

1. X is O; Y₁ is one of the moieties shown in FIG. 9 (i.e., a, b, c, ord); Y₂ is one of the moieties shown in FIG. 9 (i.e., a, b, c, or d); R₁is hydrogen, C(O)NH-Handle, C(O)-linker-Handle, C(O)NH-Acceptor,C(O)-linker-Acceptor, C(O)NH-linker-CH2-X where X is a leaving group,C(O)NH—(CH₂CH₂),C(O)N(H)-Handle where “n” is an integer ranging from 1to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” is an integer ranging from1 to 100, C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂-(CH2)₆-Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆—Cl; R₂ ishydrogen, C(O)NH-Handle, C(O)-linker-Handle, C(O)NH-Acceptor,C(O)-linker-Acceptor, C(O)NH-linker-CH2-X where X is a leaving group,C(O)NH—(CH₂CH₂)_(n)C(O)N(H)-Handle where “n” is an integer ranging from1 to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” is an integer rangingfrom 1 to 100, C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂-(CH2)₆-Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆-Cl; A₁ ishydrogen, alkyl or substituted alkyl; A₂ is hydrogen, alkyl orsubstituted alkyl; Z is O.

2. X is O; Y₁ is one of the moieties shown in FIG. 9 (i.e., a, b, c, ord); Y₂ is one of the moieties shown in FIG. 9 (i.e., a, b, c, or d); R₁is hydrogen, C(O)NH-Handle, C(O)-linker-Handle, C(O)NH-Acceptor,C(O)-linker-Acceptor, C(O)NH-linker-CH2-X where X is a leaving group,C(O)NH—(CH₂CH₂)_(n)C(O)N(H)-Handle where “n” is an integer ranging from1 to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” is an integer rangingfrom 1 to 100, C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂-(CH2)₆-Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆-Cl; R₂ ishydrogen, C(O)NH-Handle, C(O)-linker-Handle, C(O)NH-Acceptor,C(O)-linker-Acceptor, C(O)NH-linker-CH2-X where X is a leaving group,C(O)NH—(CH₂CH₂),C(O)N(H)-Handle where “n” is an integer ranging from 1to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” is an integer ranging from1 to 100, C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂-(CH2)₆-Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆-Cl; A₁ ishydrogen, alkyl or substituted alkyl; A₂ is hydrogen, alkyl orsubstituted alkyl; Z is S.

3. X is O; Y₁ is one of the moieties shown in FIG. 9 (i.e., a, b, c, ord); Y₂ is one of the moieties shown in FIG. 9 (i.e., a, b, c, or d); R₁is hydrogen, C(O)NH-Handle, C(O)-linker-Handle, C(O)NH-Acceptor,C(O)-linker-Acceptor, C(O)NH-linker-CH2-X where X is a leaving group,C(O)NH—(CH₂CH₂)_(n)C(O)N(H)-Handle where “n” is an integer ranging from1 to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” is an integer rangingfrom 1 to 100, C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂-(CH2)₆-Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆-Cl; R₂ ishydrogen, C(O)NH-Handle, C(O)-linker-Handle, C(O)NH-Acceptor,C(O)-linker-Acceptor, C(O)NH-linker-CH2-X where X is a leaving group,C(O)NH—(CH₂CH₂)_(n)C(O)N(H)-Handle where “n” is an integer ranging from1 to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” is an integer rangingfrom 1 to 100, C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂-(CH2)₆-Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆—Cl; A₁ ishydrogen, alkyl or substituted alkyl; A₂ is hydrogen, alkyl orsubstituted alkyl; Z is Se.

4. X is O; Y₁ is one of the moieties shown in FIG. 9 (i.e., a, b, c, ord); Y₂ is one of the moieties shown in FIG. 9 (i.e., a, b, c, or d); R₁is hydrogen, C(O)NH-Handle, C(O)-linker-Handle, C(O)NH-Acceptor,C(O)-linker-Acceptor, C(O)NH-linker-CH2-X where X is a leaving group,C(O)NH—(CH₂CH₂)_(n)C(O)N(H)-Handle where “n” is an integer ranging from1 to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” is an integer rangingfrom 1 to 100, C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂-(CH2)₆-Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆-Cl; R₂ ishydrogen, C(O)NH-Handle, C(O)-linker-Handle, C(O)NH-Acceptor,C(O)-linker-Acceptor, C(O)NH-linker-CH2-X where X is a leaving group,C(O)NH—(CH₂CH₂)_(n)C(O)N(H)-Handle where “n” is an integer ranging from1 to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” is an integer rangingfrom 1 to 100, C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂-(CH2)₆-Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆—Cl; A₁ ishydrogen, alkyl or substituted alkyl; A₂ is hydrogen, alkyl orsubstituted alkyl; Z is Te.

5. X is O; Y₁ is one of the moieties shown in FIG. 9 (i.e., a, b, c, ord); Y₂ is one of the moieties shown in FIG. 9 (i.e., a, b, c, or d); R₁is hydrogen, C(O)NH-Handle, C(O)-linker-Handle, C(O)NH-Acceptor,C(O)-linker-Acceptor, C(O)NH-linker-CH2-X where X is a leaving group,C(O)NH—(CH₂CH₂)_(n)C(O)N(H)-Handle where “n” is an integer ranging from1 to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” is an integer rangingfrom 1 to 100, C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂-(CH2)₆-Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆—Cl; R₂ ishydrogen, C(O)NH-Handle, C(O)-linker-Handle, C(O)NH-Acceptor,C(O)-linker-Acceptor, C(O)NH-linker-CH2-X where X is a leaving group,C(O)NH—(CH₂CH₂)_(n)C(O)N(H)-Handle where “n” is an integer ranging from1 to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” is an integer rangingfrom 1 to 100, C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂-(CH2)₆-Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆—Cl; A₁ ishydrogen, alkyl or substituted alkyl; A₂ is hydrogen, alkyl orsubstituted alkyl; Z is Acceptor.

6. X is O; Y₁ is one of the moieties shown in FIG. 9 (i.e., a, b, c, ord); Y₂ is one of the moieties shown in FIG. 9 (i.e., a, b, c, or d); R₁is hydrogen, C(O)NH-Handle, C(O)-linker-Handle, C(O)NH-Acceptor,C(O)-linker-Acceptor, C(O)NH-linker-CH2-X where X is a leaving group,C(O)NH—(CH₂CH₂)_(n)C(O)N(H)-Handle where “n” is an integer ranging from1 to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” is an integer rangingfrom 1 to 100, C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂-(CH2)₆-Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆—Cl; R₂ ishydrogen, C(O)NH-Handle, C(O)-linker-Handle, C(O)NH-Acceptor,C(O)-linker-Acceptor, C(O)NH-linker-CH2-X where X is a leaving group,C(O)NH—(CH₂CH₂)_(n)C(O)N(H)-Handle where “n” is an integer ranging from1 to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” is an integer rangingfrom 1 to 100, C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂-(CH2)₆-Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆—Cl; A₁ ishydrogen, alkyl or substituted alkyl; A₂ is hydrogen, alkyl orsubstituted alkyl; Z is Acceptor “a” shown in FIG. 8.

7. X is O; Y₁ is one of the moieties shown in FIG. 9 (i.e., a, b, c, ord); Y₂ is one of the moieties shown in FIG. 9 (i.e., a, b, c, or d); R₁is hydrogen, C(O)NH-Handle, C(O)-linker-Handle, C(O)NH-Acceptor,C(O)-linker-Acceptor, C(O)NH-linker-CH2-X where X is a leaving group,C(O)NH—(CH₂CH₂)_(n)C(O)N(H)-Handle where “n” is an integer ranging from1 to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” is an integer rangingfrom 1 to 100, C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂-(CH2)₆-Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆—Cl; R₂ ishydrogen, C(O)NH-Handle, C(O)-linker-Handle, C(O)NH-Acceptor,C(O)-linker-Acceptor, C(O)NH-linker-CH2-X where X is a leaving group,C(O)NH—(CH₂CH₂)_(n)C(O)N(H)-Handle where “n” is an integer ranging from1 to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” is an integer rangingfrom 1 to 100, C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂-(CH2)₆-Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆—Cl; A₁ ishydrogen, alkyl or substituted alkyl; A₂ is hydrogen, alkyl orsubstituted alkyl; Z is Acceptor “b” shown in FIG. 8.

8. X is O; Y₁ is one of the moieties shown in FIG. 9 (i.e., a, b, c, ord); Y₂ is one of the moieties shown in FIG. 9 (i.e., a, b, c, or d); R₁is hydrogen, C(O)NH-Handle, C(O)-linker-Handle, C(O)NH-Acceptor,C(O)-linker-Acceptor, C(O)NH-linker-CH2-X where X is a leaving group,C(O)NH—(CH₂CH₂)_(n)C(O)N(H)-Handle where “n” is an integer ranging from1 to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” is an integer rangingfrom 1 to 100, C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂-(CH2)₆-Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆—Cl; R₂ ishydrogen, C(O)NH-Handle, C(O)-linker-Handle, C(O)NH-Acceptor,C(O)-linker-Acceptor, C(O)NH-linker-CH2-X where X is a leaving group,C(O)NH—(CH₂CH₂)_(n)C(O)N(H)-Handle where “n” is an integer ranging from1 to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” is an integer rangingfrom 1 to 100, C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂-(CH2)₆-Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆—Cl; A₁ ishydrogen, alkyl or substituted alkyl; A₂ is hydrogen, alkyl orsubstituted alkyl; Z is Acceptor “c” shown in FIG. 8.

9. X is O; Y₁ is one of the moieties shown in FIG. 9 (i.e., a, b, c, ord); Y₂ is one of the moieties shown in FIG. 9 (i.e., a, b, c, or d); R₁is hydrogen, C(O)NH-Handle, C(O)-linker-Handle, C(O)NH-Acceptor,C(O)-linker-Acceptor, C(O)NH-linker-CH2-X where X is a leaving group,C(O)NH-(CH₂CH₂)_(n)C(O)N(H)-Handle where “n” is an integer ranging from1 to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” is an integer rangingfrom 1 to 100, C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂-(CH2)₆-Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆-Cl; R₂ ishydrogen, C(O)NH-Handle, C(O)-linker-Handle, C(O)NH-Acceptor,C(O)-linker-Acceptor, C(O)NH-linker-CH2-X where X is a leaving group,C(O)NH—(CH₂CH₂),C(O)N(H)-Handle where “n” is an integer ranging from 1to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” is an integer ranging from1 to 100, C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂-(CH2)₆-Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆—Cl; A₁ ishydrogen, alkyl or substituted alkyl; A₂ is hydrogen, alkyl orsubstituted alkyl; Z is Acceptor “d” shown in FIG. 8.

10. X is O; Y₁ is one of the moieties shown in FIG. 9 (i.e., a, b, c, ord); Y₂ is one of the moieties shown in FIG. 9 (i.e., a, b, c, or d); R₁is hydrogen, C(O)NH-Handle, C(O)-linker-Handle, C(O)NH-Acceptor,C(O)-linker-Acceptor, C(O)NH-linker-CH2-X where X is a leaving group,C(O)NH—(CH₂CH₂)_(n)C(O)N(H)-Handle where “n” is an integer ranging from1 to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” is an integer rangingfrom 1 to 100, C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂-(CH2)₆-Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆—Cl; R₂ ishydrogen, C(O)NH-Handle, C(O)-linker-Handle, C(O)NH-Acceptor,C(O)-linker-Acceptor, C(O)NH-linker-CH2-X where X is a leaving group,C(O)NH—(CH₂CH₂)_(n)C(O)N(H)-Handle where “n” is an integer ranging from1 to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” is an integer rangingfrom 1 to 100, C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂-(CH2)₆-Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆-Cl; A₁ ishydrogen, alkyl or substituted alkyl; A₂ is hydrogen, alkyl orsubstituted alkyl; Z is Acceptor “e” shown in FIG. 8.

11. X is O; Y₁ is one of the moieties shown in FIG. 9 (i.e., a, b, c, ord); Y₂ is one of the moieties shown in FIG. 9 (i.e., a, b, c, or d); R₁is hydrogen, C(O)NH-Handle, C(O)-linker-Handle, C(O)NH-Acceptor,C(O)-linker-Acceptor, C(O)NH-linker-CH2-X where X is a leaving group,C(O)NH—(CH₂CH₂)_(n)C(O)N(H)-Handle where “n” is an integer ranging from1 to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” is an integer rangingfrom 1 to 100, C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂-(CH2)₆-Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆—Cl; R₂ ishydrogen, C(O)NH-Handle, C(O)-linker-Handle, C(O)NH-Acceptor,C(O)-linker-Acceptor, C(O)NH-linker-CH2-X where X is a leaving group,C(O)NH—(CH₂CH₂).C(O)N(H)-Handle where “n” is an integer ranging from 1to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” is an integer ranging from1 to 100, C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂-(CH2)₆-Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆-Cl; A₁ ishydrogen, alkyl or substituted alkyl; A₂ is hydrogen, alkyl orsubstituted alkyl; Z is Acceptor “f” shown in FIG. 8.

12. X is O; Y₁ is one of the moieties shown in FIG. 9 (i.e., a, b, c, ord); Y₂ is one of the moieties shown in FIG. 9 (i.e., a, b, c, or d); R₁is hydrogen, C(O)NH-Handle, C(O)-linker-Handle, C(O)NH-Acceptor,C(O)-linker-Acceptor, C(O)NH-linker-CH2-X where X is a leaving group,C(O)NH-(CH₂CH₂),C(O)N(H)-Handle where “n” is an integer ranging from 1to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” is an integer ranging from1 to 100, C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂-(CH2)₆-Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆-Cl; R₂ ishydrogen, C(O)NH-Handle, C(O)-linker-Handle, C(O)NH-Acceptor,C(O)-linker-Acceptor, C(O)NH-linker-CH2-X where X is a leaving group,C(O)NH—(CH₂CH₂)_(n)C(O)N(H)-Handle where “n” is an integer ranging from1 to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” is an integer rangingfrom 1 to 100, C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂-(CH2)₆-Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆—Cl; A₁ ishydrogen, alkyl or substituted alkyl; A₂ is hydrogen, alkyl orsubstituted alkyl; Z is Acceptor “g” shown in FIG. 8.

13. X is O; Y₁ is one of the moieties shown in FIG. 9 (i.e., a, b, c, ord); Y₂ is one of the moieties shown in FIG. 9 (i.e., a, b, c, or d); R₁is hydrogen, C(O)NH-Handle, C(O)-linker-Handle, C(O)NH-Acceptor,C(O)-linker-Acceptor, C(O)NH-linker-CH2-X where X is a leaving group,C(O)NH—(CH₂CH₂)_(n)C(O)N(H)-Handle where “n” is an integer ranging from1 to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” is an integer rangingfrom 1 to 100, C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂-(CH2)₆-Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆—Cl; R₂ ishydrogen, C(O)NH-Handle, C(O)-linker-Handle, C(O)NH-Acceptor,C(O)-linker-Acceptor, C(O)NH-linker-CH2-X where X is a leaving group,C(O)NH—(CH₂CH₂)_(n)C(O)N(H)-Handle where “n” is an integer ranging from1 to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” is an integer rangingfrom 1 to 100, C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂-(CH2)₆-Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆-Cl; A₁ ishydrogen, alkyl or substituted alkyl; A₂ is hydrogen, alkyl orsubstituted alkyl; Z is Acceptor “h” shown in FIG. 8.

14. X is O; Y₁ is one of the moieties shown in FIG. 9 (i.e., a, b, c, ord); Y₂ is one of the moieties shown in FIG. 9 (i.e., a, b, c, or d); R₁is hydrogen, C(O)NH-Handle, C(O)-linker-Handle, C(O)NH-Acceptor,C(O)-linker-Acceptor, C(O)NH-linker-CH2-X where X is a leaving group,C(O)NH—(CH₂CH₂),C(O)N(H)-Handle where “n” is an integer ranging from 1to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” is an integer ranging from1 to 100, C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂-(CH2)₆-Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆—Cl; R₂ ishydrogen, C(O)NH-Handle, C(O)-linker-Handle, C(O)NH-Acceptor,C(O)-linker-Acceptor, C(O)NH-linker-CH2-X where X is a leaving group,C(O)NH—(CH₂CH₂)_(n)C(O)N(H)-Handle where “n” is an integer ranging from1 to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” is an integer rangingfrom 1 to 100, C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂-(CH2)₆-Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆—Cl; A₁ ishydrogen, alkyl or substituted alkyl; A₂ is hydrogen, alkyl orsubstituted alkyl; Z is Acceptor “i” shown in FIG. 8.

FIGS. 10-13 show examples of structures of compounds according to thepresent invention, where “n” is 1, 2, 3 or 4 and “HT” is(CH₂CH₂O)₂—(CH₂)₆—Cl or (CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆—Cl.

FIG. 18A shows a general structure for compounds according to thepresent invention, where the elements X, Y₁, Y₂, and R₁ are defined asfollows:

X is O, N-alkyl, S, Si(alkyl)₂ or C(alkyl)₂;

Y₁ is O, N(alkyl)₂ or one of the moieties shown in FIG. 9 (i.e., a, b,c, or d);

Y₂ is O, N(alkyl)₂ or one of the moieties shown in FIG. 9 (i.e., a, b,c, or d);

R₁ is hydrogen, C(O)NH-Handle, C(O)-linker-Handle, C(O)NH-Acceptor,C(O)-linker-Acceptor, C(O)NH-linker-CH₂-X where X is a leaving group,C(O)NH—(CH₂CH₂)_(n)C(O)N(H)-Handle where “n” is an integer ranging from1 to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” is an integer rangingfrom 1 to 100, C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH2)₆-Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆—Cl.

Nonlimiting examples of compounds according to the present invention are(in reference to FIG. 18A):

1. X is O, Y₁ is O, Y₂ is O, R₁ is hydrogen, C(O)NH-Handle,C(O)-linker-Handle, C(O)NH-Acceptor, C(O)-linker-Acceptor, orC(O)NH-linker-CH2-X where X is a leaving group.

2. X is O, Y₁ is N(alkyl)₂, Y₂ is N(alkyl)₂, R₁ is hydrogen,C(O)NH-Handle, C(O)-linker-Handle, C(O)NH-Acceptor,C(O)-linker-Acceptor, or C(O)NH-linker-CH₂-X where X is a leaving group.

3. X is O, Y₁ is moiety “a” in FIG. 9, Y₂ is moiety “a” in FIG. 9, R₁ ishydrogen, C(O)NH-Handle, C(O)-linker-Handle, C(O)NH-Acceptor,C(O)-linker-Acceptor, or C(O)NH-linker-CH₂-X where X is a leaving group.

FIG. 18B shows a general structure for compounds according to thepresent invention, where the elements X, R₁₀-R₁₉, A₁, A₂ and Z aredefined as follows:

X is O, N-alkyl, S, Si(alkyl)₂ or C(alkyl)₂;

R₁₀ and R₁₇ are independently selected from —H, alkyl (e.g., methyl,ethyl, propyl, butyl, etc.), —CH₂—SO₃H, —CH₂CH₂—SO₃H, —CH₂OCH₂—SO₃H,—CH₂CH₂OCH₂CH₂—SO₃H, —CH₂—CO₂H, —CH₂—CH₂—CO₂H, —CH₂OCH₂—CO₂H,—CH₂CH₂OCH₂CH₂—CO₂H;

R₁₁, R₁₂, R₁₅ and R₁₆ are independently selected from H, alkyl (e.g.,methyl, ethyl, propyl, butyl, etc.), —CH₂—SO₃H, —CH₂CH₂—SO₃H,—CH₂OCH₂—SO₃H, —CH₂CH₂OCH₂CH₂—SO₃H, —CH₂—CO₂H, —CH₂—CH₂—CO₂H,—CH₂OCH₂—CO₂H, —CH₂CH₂OCH₂CH₂—CO₂H;

R₁₃ and R₁₄ are independently selected from alkyl (e.g., methyl, ethyl,propyl, butyl, etc.), —C(O)CH₃, —C(O)CH₂CH₃, —C(O)OCH₃, —C(O)OCH₂CH₃,—C(O)N(CH₃)₂, —C(O)N(CH₂CH₃)₂;

R₁₈ is hydrogen, C(O)NH-Handle, C(O)-linker-Handle, C(O)NH-Acceptor,C(O)-linker-Acceptor, C(O)NH-linker-CH2-X where X is a leaving group,C(O)NH—(CH₂CH₂)_(n)C(O)N(H)-Handle where “n” is an integer ranging from1 to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” is an integer rangingfrom 1 to 100, C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂-(CH2)₆-Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆—Cl;

R₁₉ is hydrogen, C(O)NH-Handle, C(O)-linker-Handle, C(O)NH-Acceptor,C(O)-linker-Acceptor, C(O)NH-linker-CH2-X where X is a leaving group,C(O)NH—(CH₂CH₂)_(n)C(O)N(H)-Handle where “n” is an integer ranging from1 to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” is an integer rangingfrom 1 to 100, C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂-(CH2)₆-Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆—Cl;

A₁ is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl,aromatic, heteroaromatic, substituted alkyl, substituted alkenyl,substituted alkynyl, substituted heteroalkyl, substituted heteroalkenyl,heterocycloalkyl, heterocycloalkenyl, substituted aromatic group, orsubstituted heteroaromatic group;

A₂ is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl,aromatic, heteroaromatic, substituted alkyl, substituted alkenyl,substituted alkynyl, substituted heteroalkyl, substituted heteroalkenyl,heterocycloalkyl, heterocycloalkenyl, substituted aromatic group, orsubstituted heteroaromatic group;

Z is O, S, Se, Te or an Acceptor.

Nonlimiting examples of compounds according to the present invention are(in reference to FIG. 18B):

1. X is O; R₁₀ and R₁₇ are independently selected from —H, methyl, and—CH₂—SO₃H; R₁₁, R_(12,) R₁₅ and R₁₆ are independently selected from Hand methyl; R₁₃ and R₁₄ are independently selected from methyl, ethyl,and —C(O)CH₃; R₁₈ is hydrogen, C(O)NH-Handle, C(O)-linker-Handle,C(O)NH-Acceptor, C(O)-linker-Acceptor, or C(O)NH-linker-CH2-X where X isa leaving group; R₁₉ is hydrogen, C(O)NH-Handle, C(O)-linker-Handle,C(O)NH-Acceptor, C(O)-linker-Acceptor, or C(O)NH-linker-CH2-X where X isa leaving group; A₁ is hydrogen, alkyl or substituted alkyl; A₂ ishydrogen, alkyl or substituted alkyl; Z is O.

2. X is O; R₁₀ and R₁₇ are independently selected from —CH₂—SO₃H; R₁₁,R_(12,) R₁₅ and R₁₆ are methyl; R₁₃ and R₁₄ are methyl; R₁₈ is hydrogen,C(O)NH-Handle, C(O)-linker-Handle, C(O)NH-Acceptor,C(O)-linker-Acceptor, or C(O)NH-linker-CH₂-X where X is a leaving group;R₁₉ is hydrogen, C(O)NH-Handle, C(O)-linker-Handle, C(O)NH-Acceptor,C(O)-linker-Acceptor, or C(O)NH-linker-CH₂-X where X is a leaving group;A₁ is hydrogen, alkyl or substituted alkyl; A₂ is hydrogen, alkyl orsubstituted alkyl; Z is O.

FIG. 19 shows a general structure for compounds according to the presentinvention, where the elements X, R₂₀, R₂₁, A₁, A₂ and Z are defined asfollows:

X is O, N-alkyl, S, Si(alkyl)₂ or C(alkyl)₂;

R₂₀ is hydrogen, C(O)NH-Handle, C(O)-linker-Handle, C(O)NH-Acceptor,C(O)-linker-Acceptor, C(O)NH-linker-CH2-X where X is a leaving group,C(O)NH—(CH₂CH₂)_(n)C(O)N(H)-Handle where “n” is an integer ranging from1 to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” is an integer rangingfrom 1 to 100, C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂-(CH2)₆-Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆-Cl;

R₂₁ is hydrogen, C(O)NH-Handle, C(O)-linker-Handle, C(O)NH-Acceptor,C(O)-linker-Acceptor, C(O)NH-linker-CH2-X where X is a leaving group,C(O)NH—(CH₂CH₂)_(n)C(O)N(H)-Handle where “n” is an integer ranging from1 to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” is an integer rangingfrom 1 to 100, C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂-(CH2)₆-Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆—Cl;

A₁ is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl,aromatic, heteroaromatic, substituted alkyl, substituted alkenyl,substituted alkynyl, substituted heteroalkyl, substituted heteroalkenyl,heterocycloalkyl, heterocycloalkenyl, substituted aromatic group, orsubstituted heteroaromatic group;

A₂ is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl,aromatic, heteroaromatic, substituted alkyl, substituted alkenyl,substituted alkynyl, substituted heteroalkyl, substituted heteroalkenyl,heterocycloalkyl, heterocycloalkenyl, substituted aromatic group, orsubstituted heteroaromatic group;

Z is O, S, Se, Te or an Acceptor.

Nonlimiting examples of compounds according to the present invention are(in reference to FIG. 19):

1. X is O; R₂₀ is hydrogen, C(O)NH-Handle, C(O)-linker-Handle,C(O)NH-Acceptor, C(O)-linker-Acceptor, or C(O)NH-linker-CH₂-X where X isa leaving group; R₂₁ is hydrogen, C(O)NH-Handle, C(O)-linker-Handle,C(O)NH-Acceptor, C(O)-linker-Acceptor, or C(O)NH-linker-CH₂-X where X isa leaving group; A₁ is hydrogen, alkyl or substituted alkyl; A₂ ishydrogen, alkyl or substituted alkyl; Z is O.

2. X is O; R₂₀ is hydrogen, C(O)NH-Handle, C(O)-linker-Handle,C(O)NH-Acceptor, C(O)-linker-Acceptor, or C(O)NH-linker-CH₂-X where X isa leaving group; R₂₁ is hydrogen, C(O)NH-Handle, C(O)-linker-Handle,C(O)NH-Acceptor, C(O)-linker-Acceptor, or C(O)NH-linker-CH₂-X where X isa leaving group; A₁ is hydrogen or alkyl; A₂ is hydrogen or alkyl; Z isO.

FIG. 20A shows a general structure for compounds according to thepresent invention, where the elements, where the elements X, Y₁, Y₂, R₁,R₂, R₃, R₄, R₅, R₆, A₁ and A₂ are defined as follows:

X is O, N-alkyl, S, Si(alkyl)₂ or C(alkyl)₂;

Y₁ is O, OH, NH₂, N(alkyl)₂ or one of the moieties shown in FIG. 9(i.e., a, b, c, or d);

Y₂ is O, OH, NH₂, N(alkyl)₂ or one of the moieties shown in FIG. 9(i.e., a, b, c, or d);

R₁ (which can be a substitution at either the 5′ position, the 6′position or both) is hydrogen, C(O)NH-Handle, C(O)-linker-Handle,C(O)NH-Acceptor, C(O)-linker-Acceptor, C(O)NH-linker-CH2-X where X is aleaving group, C(O)NH—(CH₂CH₂)_(n)C(O)N(H)-Handle where “n” is aninteger ranging from 1 to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” isan integer ranging from 1 to 100,C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂-(CH2)₆-Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆—Cl;

R₂ is hydrogen, C(O)NH-Handle, C(O)-linker-Handle, C(O)NH-Acceptor,C(O)-linker-Acceptor, C(O)NH-linker-CH2-X where X is a leaving group,C(O)NH—(CH₂CH₂)_(n)C(O)N(H)-Handle where “n” is an integer ranging from1 to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” is an integer rangingfrom 1 to 100, C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂-(CH2)₆-Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆—Cl;

R₃, R₄, R₅ and R₆ are independently hydrogen, alkyl, —SO₃H, halogen, orR₃ and Y₁ can form a ring, or R₄ and Y₁ can form a ring, or R₅ and Y₂can form a ring, or R₆ and Y₂ can form a ring;

A₁ is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl,aromatic, heteroaromatic, substituted alkyl, substituted alkenyl,substituted alkynyl, substituted heteroalkyl, substituted heteroalkenyl,heterocycloalkyl, heterocycloalkenyl, substituted aromatic group, orsubstituted heteroaromatic group;

A₂ is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl,aromatic, heteroaromatic, substituted alkyl, substituted alkenyl,substituted alkynyl, substituted heteroalkyl, substituted heteroalkenyl,heterocycloalkyl, heterocycloalkenyl, substituted aromatic group, orsubstituted heteroaromatic group.

FIG. 20B shows a general structure for compounds according to thepresent invention, where the elements R₁ and R₂ are defined as follows:

R₁ (which can be a substitution at either the 5′ position, the 6′position or both) is hydrogen, C(O)NH-Handle, C(O)-linker-Handle,C(O)NH-Acceptor, C(O)-linker-Acceptor, C(O)NH-linker-CH2-X where X is aleaving group, C(O)NH—(CH₂CH₂)_(n)C(O)N(H)-Handle where “n” is aninteger ranging from 1 to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” isan integer ranging from 1 to 100,C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂-(CH2)₆-Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆—Cl;

R₂ is hydrogen, C(O)NH-Handle, C(O)-linker-Handle, C(O)NH-Acceptor,C(O)-linker-Acceptor, C(O)NH-linker-CH2-X where X is a leaving group,C(O)NH—(CH₂CH₂)_(n)C(O)N(H)-Handle where “n” is an integer ranging from1 to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” is an integer rangingfrom 1 to 100, C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂-(CH2)₆-Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆—Cl;

FIG. 20C shows a general structure for compounds according to thepresent invention, where the elements R₁ and R₂ are defined as follows:

R₁ (which can be a substitution at either the 5′ position, the 6′position or both) is hydrogen, C(O)NH-Handle, C(O)-linker-Handle,C(O)NH-Acceptor, C(O)-linker-Acceptor, C(O)NH-linker-CH2-X where X is aleaving group, C(O)NH—(CH₂CH₂)_(n)C(O)N(H)-Handle where “n” is aninteger ranging from 1 to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” isan integer ranging from 1 to 100,C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂-(CH2)₆-Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆—Cl;

R₂ is hydrogen, C(O)NH-Handle, C(O)-linker-Handle, C(O)NH-Acceptor,C(O)-linker-Acceptor, C(O)NH-linker-CH2-X where X is a leaving group,C(O)NH—(CH₂CH₂)_(n)C(O)N(H)-Handle where “n” is an integer ranging from1 to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” is an integer rangingfrom 1 to 100, C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂-(CH2)₆-Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆—Cl;

Any suitable synthetic method and/or scheme can be used to synthesizecompounds according to the present invention. General synthetic methodsfor the synthesis of rhodamine dyes and derivatives are presented inBeija et. al. “Synthesis and Applications of Rhodamine Derivatives asFluorescent Probes”, Chem Soc Rev 2009, 38, 2410-2433, which isincorporated-by-reference into this document for all purposes. Generalsynthetic methods for the synthesis of coumarin units are presented inVekariva et al. “Recent Advances in the Synthesis of CoumarinDerivatives via Knoevenagel Condensation: A Review”, Syn Comm 2014, 44,2756-2788, which is incorporated-by-reference into this document for allpurposes. Various tagging methods are discussed above includingreferences related to synthesizing and using the tags. Synthetic schemesare shown, for example, in FIGS. 15-17 and 21-27.

Compounds according to the present invention make it possible to producesuper-resolution images of subcellular structures in live cells. FIG. 14shows a super-resolution image of a nucleus using Photo-activatedLocalization Microscopy (“PALM”). To produce the image, cells werelabeled with a compound according to the present invention. Two beams oflight—one generating continuous illumination, while the other ispulsed—are used in the imaging. The compounds flicker and produce theshown image without the need for chemical additives, buffers or uncagingmethods. General methods for super-resolution imaging are presented inGodin et al. “Super-Resolution Microscopy Approaches for Live CellImaging”, Biophys J. 2014, 107, 1777-1784, which isincorporated-by-reference into this document for all purposes.

FIG. 28 shows a super-resolution image of live U2OS cells expressing H2Band labeled with PC-JF549-HT. It further shows the overlay with adiffraction limited fluorescence image with H2B expressing JF635-HT witha mean localization precision of 37 nm. FIG. 29 shows a super-resolutionimage of mitochondria expressing TOMM20 and labeled with PC-JF549-HT.The mean localization precision is 28 nm. FIG. 30 shows super-resolutioniPALM images of actin in ptk2 cells transfected with lifeact-halo, andlabeled with the PC-JF549-HT. FIG. 31 shows super-resolution images oftubulin in ptk2 cells expressing HaloTag and labeled with thePC-JF549-HT. FIG. 32 shows super-resolution iPALM images of tubulin inCOS7 cells targeted with an antibody which is labeled with PC-AF594-NHS.FIG. 33 shows super-resolution iPALM images of tubulin in COS7 cellstargeted with an antibody which is labeled with PC-AF594-NHS.

Experimental Symbols and Abbreviations

AF AlexaFluor ® Azep Azepane BG-NH₂ 6-((4-(aminomethyl)benzypoxy)-9H-purin-2-amine C₆D₆ Deuterated benzene CAM Ceric ammonium molybdateCbz-NH₂ Benzyl carbamate CDCl₃ Deuterated chloroform CD₃CN Deuteratedacetonirtile conc. Concentrated CsCO₃ Cesium carbonate DCCDicyclohexylcarbodiimide DCM Dichloromethane DIEAN,N-Diisopropylethylamine DMAP 4-Dimethylaminopyridine DMFN,N-Dimethylformamide DSC N,N′-Disuccinimidyl carbonate EDC3-(Ethyliminomethyleneamino)-N,N- dimethylpropan-1-amine ESIElectrospray Ionization Et₃N Triethylamine EtOAc Ethyl Acetate h HoursH₂O Water H₂SO₄ Sulfuric acid HATU 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate HCLHydrochloric acid HOBT Hydroxybenzotriazole HPLC High Performance LiquidChromatography Hz Hertz JF JaneliaFluor ® LC/MS Liquidchromatography/Mass spectrometry μL Microliter MeCN Acetonitrile MeOD-d₄Deuterated methanol MeOH Methanol mg Milligram mL Milliliter mmmillimeter mmol Millimol min Minutes nm Nanometer Na₂SO₄ Sodium SulfateNaOH Sodium Hydroxide NaOMe Sodium methoxide NHS N-HydroxysuccinimideNMR Nuclear Magnetic Resonance PC Photochromic Pd/C Palladium overcarbon Pd₂(dba)₃ Tris(dibenzylideneacetone)dipalladium(0) PEGPolyethelene glycol ppm Part per million quant Quantitative s Seconds RTRoom temperature TFA Trifluoroacetic acid TLC Thin layer chromatorgraphyTMSCH₂N₂ Diazomethane TMS Tetramethylsilane TSTUN,N,N′,N′-Tetramethyl-O- (N-succinimidyl)uronium tetrafluoroborate TEATriethyl amine THF Tetrahydrofuran TFA Trifluoroacetic acid UVUltraviolet Xantphos 4,5-Bis(diphenylphosphino)-9,9- dimethylxantheneXPhos 2-Dicyclohexylphosphino-2′,4′,6′- triisopropylbiphenyl % v/vVolume/volume percent % wt/wt Weight/weight percent ° C. Degrees Celsius

I. Materials and Methods

All starting materials were obtained from commercial sources and wereused as received without further purification. Anhydrous solvents, inseptum-sealed bottles, were used for all chemical reactions, which wereconducted either under ambient conditions in a 4-dram glass vial orround bottomed flask or, when specified, under argon atmosphere in asealed septum-capped vial. The reaction progress was monitored by TLCchromatography on pre-coated TLC glass plates (silica gel 60 F₂₅₄, 250μm thickness) and TLC chromatograms were visualized by a UV lamp ordeveloped with CAM, permanganate or iodine stains. When possible, LC/MS(4.6 mm×150 mm 5 μm C18 column; 5 μL injection; 10-95% MeCN/H₂O, lineargradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/minflow; ESI; positive ion mode; UV detection at 254 nm and 550 nm) wasalso used to check reaction progress.

Purification of organic molecules was performed via silica gelchromatography on an automated purification system using pre-packedsilica gel columns, or via reverse phase HPLC (10-95%, 20-80% or 30-70%MeCN/H₂O, linear gradient, with constant 0.1% v/v TFA additive; 23 minrun, 42 mL/min flow, detection at 254 nm, 500 nm, 550 nm and 650 nm.Purity was confirmed by analytical HPLC (4.6 mm×150 mm 5 μm C18 column;5 μL injection; 10-95% CH₃CN/H₂O linear gradient with constant 0.1% v/vTFA additive; 20 min run; 1 mL/min flow; ESI, positive ion mode,detection at 254 nm, 500 nm, 550 nm and 650 nm.

NMR spectra were recorded on a 400 MHz spectrometer. ¹H and ¹³C chemicalshifts (δ) were referenced to TMS or residual solvent peaks. Data for ¹HNMR spectra are reported as follows: chemical shift (δ ppm),multiplicity (s=singlet, d=doublet, t=triplet, q=quartet, dd=doublet ofdoublets, m=multiplet), coupling constant (Hz), integration. Data for¹³C NMR spectra are reported by chemical shift (δ ppm).

1. Synthesis of the Photochromic JaneliaFluor Dyes (Scheme 1, FIGS.21-22)

All the dyes presented in Scheme 1, are synthesized from two advancedintermediates (S1 and S2) whose synthesis have been described previouslyby Grimm et al.¹ From these intermediates, 7 different photochromicJaneliaFluor dyes have been synthesized, all equipped with a coumarinswitch—responsible for the photochromic behavior, and a biologicalhandle to allow for the specific targeting of proteins-of-interest. Totarget self-labeling enzymes such as HaloTag² and SNAPTag,³ three typesof linkers (HTL, exHTL,⁴ PG-NH₂ in Scheme 1 inset) were covalentlyattached to the PC-JaneliaFluor dyes. In addition, NHS and taxolvariants of these PC-JaneliaFluor dyes where prepared to allow forantibody, and tubulin labeling, respectively. Although not shown here,other biological handles such as azides, alkynes and tetrazenes forclick-chemistry or biotin can be covalently introduced into themolecular structure of these dyes to increase the range of applicationsof these probes.

Depending on the nature of the heteroatom on the rhodamine (X in Scheme1), different synthetic strategies where employed to couple the7-amino-4-(trifluoromethyl)-coumarin to the rhodamine. In polar solventssuch as DMF, rhodamines (X═O) are present in their “open” quinoidalzwitterionic form, exposing the carboxylate group at the 3-position andrendering it susceptible to activation and subsequent amidation. Thus incases where the molecular structure features two reactive carboxylatemoieties, such as intermediate S3, care needs to be taken to achieveselective activation/amidation.

On the contrary, silicorhodamines (X═Si(CH₃)₂) tend to be present in theclosed form, effectively reducing the reactivity of the carboxylate atthe 3′-position, and its susceptibility to activation/amidation undermild coupling conditions, example HATU. However, under harsh couplingconditions, for example oxalyl chloride, the silicorhodamine will opento the quinoidal form which will allow for the coupling to take place atthe 3′-position.

In order to synthesize the HaloTag (PC-JF549-HT) and extended HaloTag(PC-JF549-exHT) version of PC-JaneliaFluor 549, Scheme 1, the methylester at the 6′-position in S1 was hydrolyzed under basic conditions toreveal the carboxylate, S3, which was activated with DCC to result inthe activated NHS ester which was subsequently reacted with HTL, S4, orexHTL, S5, in the presence of excess Hünig's base to attach thebiological handle. To introduce the switch at the 3′-position, thecarboxylate was activated by HATU and 7-amino-4-(trifluoromethyl)coumarin to yield the desired fluorophore. It is beneficial to note thatthe same synthetic targets were also attained, with similar yields, byfirst coupling the 7-amino-4-(trifluoromethyl) coumarin to Si, followedby the hydrolyses of the methyl ester and the attachment of thebiological handle. This particular path was taken to synthesize both theSNAP variant, PC-JF549-SNAP and the NHS variant, PC-JF549-NHS, viaintermediate S6. To synthesize the docetaxyl variant PC-JF549-Tx, theactivated NHS ester was first coupled with 7-aminooctanoic acid, whichwill act as a spacer between the rhodamine and docetaxyl, to produceintermediate S8 which was subsequently activated and coupled with theactivated docetaxel, Tx in Scheme 1 inset, to get the desiredfluorophore.

In the case of photochromic silicorhodamine, S2 was activated withoxalyl chloride to produce the acyl chloride which was then reacted with7-amino-4-(trifluoromethyl) coumarin in the presence of excess Hünig'sbase to yield intermediate S7, which was then hydrolyzed under basiccondition, and then activated with HATU and coupled with the respectiveamine to get the HaloTag and SNAP tag versions, PC-JF646-HT andPC-JF646-SNAP, respectively.

2. General Experimental Information for Synthesis of Compounds forScheme 1

3′,6′-di(azetidin-1-yl)-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3-oxo-2-(2-oxo-4-(trifluoromethyl)-2H-chromen-7-yl)spiro[isoindoline-1,9′-xanthene]-6-carboxamide(PC-JF549-HT): A vial was charged with S4¹ (16 mg, 0.024 mmol),7-amino-4-(trifluoromethyl) coumarin (11 mg, 0.048 mmol, 2 eq.), HATU(28 mg, 0.073 mmol, 3 eq.), DIEA (42 μL, 0.24 mmol, 10 eq.) and DMF (300μL). Contents were stirred at room temperature and ambient atmospherefor 24 h, after which the solvent was concentrated to dryness, and thecrude product was purified by silica gel chromatography (0-30%EtOAc/DCM, linear gradient) to afford PC-JF549-HT (8 mg, 38%). LRMS(ESI) calcd for C₄₇H₄₆ClF₃N₄O₇ [M]⁺ 871.4, found 871.4

2-(2-(2-(3′,6′-di(azetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthene]-6-carboxamido)ethoxy)ethoxy)ethyl(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamate (S5): A vial wascharged with S3¹ (10 mg, 0.015 mmol), DSC (11 mg, 0.044 mmol, 3.0 eq.),DMAP (0.18 mg, 0.001 mmol, 0.1 eq.), DIEA (26 μL, 0.15 mmol, 10 eq.) andDMF (300 μL), and contents were stirred at room temperature and ambientatmosphere for 1 hour before exHTL⁴ (23 mg, 0.044 mmol, 3 eq.) was addedand the resultant mixture was stirred for an additional 24 h. Thesolvent was concentrated to dryness, and the crude product was purifiedby silica gel chromatography (0-10% MeOH/DCM, linear gradient) to affordS5 (7.4 mg, 61%).¹H NMR (CDCl₃, 400 MHz) δ8.03 (s, 2H), 7.56 (s, 1H),6.89 (s1H), 6.54 (d, J=8.6 Hz, 2H), 6.20 (d, J=2.3 Hz, 2H), 6.09 (dd,J=8.6, 2.3 Hz, 2H), 5.23 (s, 1H), 4.12 (t, J=4.8 Hz, 2H), 3.91 (t, J=7.3Hz, 8H), 3.65-3.50 (m, 16H), 3.49 (s, 2H), 3.44 (t, J=6.8 Hz, 2H), 3.33(q, J=5.3 Hz, 2H), 2.38 (p, J=7.2 Hz, 4H), 1.83-1.71 (m, 2H), 1.59 (p,J=6.8 Hz, 2H), 1.45 (p, J=6.7 Hz, 2H). 1.36 (m, 2H).

2-(2-(2-(3′,6′-di(azetidin-1-yl)-3-oxo-2-(2-oxo-4-(trifluoromethyl)-2H-chromen-7-yl)spiro[isoindoline-1,9′-xanthene]-6-carboxamido)ethoxy)ethoxy)ethyl(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamate (PC-JF549-ExHT): A vialwas charged with S5 (9 mg, 0.011 mmol), 7-amino-4-(trifluoromethyl)coumarin (7 mg, 0.033 mmol, 3 eq.), HATU (12 mg, 0.033 mmol, 3 eq.),DIEA (19 μL, 0.11 mmol, 10 eq.) and DMF (300 μL). Contents were stirredat room temperature and ambient atmosphere for 24 h, after which thesolvent was concentrated to dryness, and the crude product was purifiedby silica gel chromatography (0-5% MeOH/DCM, linear gradient) to affordPC-JF549-exHT (2 mg, 18%),¹H NMR (CDCl₃, 400 MHz) δ8.05 (d, J=7.9 Hz,1H), 7.92 (d, J=8.0 Hz, 1H), 7.49 (m, 2H), 7.30 (dd, J=8.9, 2.1 Hz, 1H),7.17 (d, J=2.1 Hz, 1H), 6.79 (s, 1H), 6.65 (s, 1H), 6.57 (d, J=8.6 Hz,2H), 6.12 (d, J=2.3 Hz, 2H), 6.02 (dd, J=8.6, 2.3 Hz, 2H), 5.23 (s, 1H),4.14 (s, 2H), 3.89 (t, J=7.3 Hz, 8H), 3.67-3.50 (m, 18H), 3.45 (t, J=6.6Hz, 2H), 3.33 (d, J=5.6 Hz, 2H), 2.37 (p, J=7.2 Hz, 4H), 1.77 (p, J=6.7Hz, 2H), 1.60 (d, J=14.6 Hz, 2H), 1.44 (q, J=8.1, 6.8 Hz, 2H), 1.37 (d,J=7.0 Hz).

methyl3′,6′-di(azetidin-1-yl)-3-oxo-2-(2-oxo-4-(trfluoromethyl)-2H-chromen-7-yl)spiro[isoindoline-1,9′-xanthene]-6-carboxylate(S6): A vial was charged with S1¹ (50 mg, 0.107 mmol),7-amino-4-(trifluoromethyl) coumarin (73 mg, 0.320 mmol, 3 eq.), HATU(122 mg, 0.320 mmol, 3.0 eq.), DIEA (200 μL, 1.07 mmol, 10 eq.) and DMF(1.00 mL). Contents were stirred at room temperature and ambientatmosphere for 24 h, after which the solvent was concentrated todryness, and the crude product was purified by silica gel chromatography(0-30% EtOAc/DCM, linear gradient) to afford S6 (45 mg, 62%). ¹H NMR(CDCl₃, 400 MHz) δ8.16 (dd, J=8.0, 1.4 Hz, 1H), 8.06 (dd, J=8.0, 0.7 Hz,1H), 7.69 (dd, J=1.3, 0.7 Hz, 1H), 7.49 (dq, J=8.9, 1.9 Hz, 1H), 7.29(dd, J=8.9, 2.2 Hz, 1H), 7.16 (d, J=2.1 Hz, 1H), 6.65 (d, J=0.9 Hz, 1H),6.57 (d, J=8.6 Hz, 2H), 6.13(d, J=2.3 Hz, 2H), 6.03 (dd, J=8.6, 2.3 Hz,2H), 3.89 (t, J=7.3 Hz, 8H), 3.85 (s, 3H), 2.37 (p, J=7.3 Hz, 4H).

methyl3,7-di(azetidin-1-yl)-5,5-dimethyl-3′-oxo-2′-(2-oxo-4-(trifluoromethyl)-2H-chromen-7-yl)-5H-spiro[dibenzo[b,e]siline-10,1′-isoindoline]-6′-carboxylate(S7): A vial was charged with S2¹ (60 mg, 0.117 mmol), oxalyl chloride(12 μL, 0.141 mmol, 1.2 eq.) and DCM (1.00 mL) and the content wasstirred at room temperature and ambient atmosphere for 30 minutes.7-amino-4-(trifluoromethyl) coumarin (81 mg, 0.352 mmol, 3 eq.) and DIEA(126 μL, 0.704 mmol, 6 eq.) were then added and the contents werestirred for 1 h, after which the solvent was concentrated to dryness,and the crude product was purified by silica gel chromatography (0-30%EtOAc/DCM, linear gradient) to afford S7 (53 mg, 62%). ¹H NMR (CDCl₃,400 MHz) δ8.09-7.95 (m, 2H), 7.78 (m, 1H), 7.60 (td, J=4.6, 2.2 Hz, 1H),7.54 (tt, J=5.6, 1.1 Hz, 1H), 7.45 (m, 1H), 6.86 (d, J=2.6 Hz, 1H), 6.76(ddd, J=8.8, 3.7, 1.2 Hz, 2H), 6.62 (d, J=2.8 Hz, 1H), 6.60 (s, 1H),6.42 (dd, J=8.9, 2.7 Hz, 1H), 6.24 (dd, J=8.8, 2.6 Hz, 1H), 3.88 (t,J=7.2 Hz, 4H), 3.81 (s, 3H), 3.65 (t, J=6.2 Hz, 2H), 3.31 (t, J=6.6 Hz,2H), 2.35 (p, J=7.2 Hz, 2H), 2.06 (p, J=6.3 Hz, 2H), 0.71 (s, 3H), 0.59(s, 3H).

3,7-di(azetidin-1-yl)-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-5,5-dimethyl-3′-oxo-2′-(2-oxo-4-(trifluoromethyl)-2H-chromen-7-yl)-5H-spiro[dibenzo[b,e]siline-10,1′-isoindoline]-6′-carboxamide(PC-JF646-HT): A vial was charged with S7 (53 mg, 0.073 mmol), NaOH (734μL, 1M, 10 eq.) MeOH (5.0 mL) and THF (2.5 mL). Content was stirred atroom temperature and ambient atmosphere for 24 h, after which HCl (770μL, 1M, 10.5 eq.) was added to quench the reaction. The organic layerwas extracted with DCM (3×10 mL). Organic layers combined, dried(Na₂SO₄), and concentrated. The hydrolyzed residue was carried to thenext step without further purification. A vial was charged withhydrolyzed S7 (20 mg, 0.028 mmol), HTL (28 mg, 0.085 mmol, 3 eq.), HATU(32 mg, 0.085 mmol, 3.0 eq.), DIEA (49 pt, 0.28 mmol, 10 eq.) and DMF(300 μL). Contents were stirred at room temperature and ambientatmosphere for 24 h, after which the solvent was concentrated todryness, and the crude product was purified by silica gel chromatography(0-40% EtOAc/DCM, linear gradient) to afford PC-JF646-HT (10.4 mg, 40%).¹H NMR (CDCl₃, 400 MHz) δ8.00 (d, J=7.8 Hz, 1H), 7.72 (dd, J=9.1, 2.3Hz, 1H), 7.69 (dd, J=7.9, 1.5 Hz, 1H), 7.61 (d, J=2.3 Hz, 1H), 7.43 (dd,J=9.1, 2.0 Hz, 1H), 7.30 (d, J=1.2 Hz, 1H), 6.76 (d, J=8.8 Hz, 2H), 6.62(d, J=4.8 Hz, 1H), 6.61 (d, J=2.7 Hz, 2H), 6.59 (s, 1H), 6.24 (d, J=2.6Hz, 1H), 6.22 (d, J=2.7 Hz, 1H), 3.88 (t, J=7.3 Hz, 8H), 3.64-3.54 (m,8H), 3.51 (t, J=6.7 Hz, 2H), 3.41 (t, J=6.7 Hz, 2H), 2.35 (p, J=7.2 Hz,4H), 1.79-1.69 (m, 2H), 1.58-1.50 (m, 2H), 1.47-1.37 (m, 2H), 1.37-1.27(m, 2H), 0.70 (s, 3H), 0.57 (s, 3H).

N-(4-(((2-amino-9H-purin-6-yl)oxy)methyl)benzyl)-3,7-di(azetidin-1-yl)-5,5-dimethyl-3′-oxo-2′-(2-oxo-4-(trifluoromethyl)-2H-chromen-7-yl)-5H-spiro[dibenzo[b,e]siline-10,1′-isoindoline]-6′-carboxamide(PC-JF549-SNAP): A vial was charged with hydrolyzed S7 (18 mg, 0.025mmol), BG-NH2 (21 mg, 0.076 mmol, 3 eq.), HATU (29 mg, 0.076 mmol, 3.0eq.), DIEA (44 μL, 0.25 mmol, 10 eq.) and DMF (300 μL). Contents werestirred at room temperature and ambient atmosphere for 24 h, after whichthe solvent was concentrated to dryness, and the crude product waspurified by silica gel chromatography (0-10% MeOH/DCM, linear gradient)to afford PC-JF646-SNAP (11 mg, 45%). LRMS (ESI) calcd forC₅₂H₄₄F3N₉O₅Si [M]⁺ 960.1, found 960.4

N-(4-(((2-amino-9H-purin-6-yl)oxy)methyl)benzyl)-3′,6′-di(azetidin-1-yl)-3-oxo-2-(2-oxo-4-(trifluoromethyl)-2H-chromen-7-yl)spiro[isoindoline-1,9′-xanthene]-6-carboxamide(PC-JF549-SNAP): A vial was charged with S6 (70 mg, 0.103 mmol), NaOH(1.03 mL, 1M, 10 eq.) MeOH (5.0 mL) and THF (2.5 mL). Content wasstirred at room temperature and ambient atmosphere for 24 h, after whichHCl (1.08 mL, 1M, 10.5 eq.) was added to quench the reaction. Theorganic layer was extracted with DCM (3×10 mL). Organic layers combined,dried (Na₂SO₄), and concentrated. The hydrolyzed residue was carried tothe next step without further purification. A vial was charged withhydrolyzed S6 (10 mg, 0.015 mmol), BG-NH2 (12 mg, 0.045 mmol, 3 eq.),HATU (17 mg, 0.045 mmol, 3.0 eq.), DIEA (26 μL, 0.15 mmol, 10 eq.) andDMF (300 μL). Contents were stirred at room temperature and ambientatmosphere for 24 h, after which the solvent was concentrated todryness, and the crude product was purified by silica gel chromatography(0-40% EtOAc/DCM, linear gradient) to afford PC-JF549-SNAP (12 mg, 87%).¹H NMR (CDCl₃, 400 MHz) δ8.55 (s, 1H), 8.24 (d, J=8.4 Hz, 1H), 8.05 (dd,J=8.0, 1.4 Hz, 1H), 8.00 (d, J=7.9 Hz, 1H), 7.77 (t, J=5.6 Hz, 1H), 7.69(s, 1H), 7.52 (d, J=3.7 Hz, 1H), 7.37 (d, J=7.8 Hz, 1H), 7.31 (s, J=7.2Hz, 2H), 7.23 (d, J=7.9 Hz, 2H), 6.93 (t, J=8.0 Hz, 1H), 6.57 (d, J=8.5Hz, 2H), 6.54 (dd, J=8.0, 1.4 Hz, 1H), 6.40 (td, J=7.7, 7.3, 1.4 Hz,1H), 6.03 (d, J=8.7 Hz, 2H), 5.99 (m, 3H), 5.38 (s, 2H), 4.49 (s, 2H),3.84 (h, J=7.3 Hz, 8H), 2.34 (p, J=7.3 Hz, 4H).

2,5-dioxopyrrolidin-1-yl3′,6′-di(azetidin-1-yl)-3-oxo-2-(2-oxo-4-(trifluoromethyl)-2H-chromen-7-yl)spiro[isoindoline-1,9′-xanthene]-6-carboxylate(PC-JF549-NHS): A vial was charged with S6 (45 mg, 0.066 mmol), NaOH(660 μL, 1M, 10 eq.) MeOH (5.0 mL) and THF (2.5 mL). Content was stirredat room temperature and ambient atmosphere for 24 h, after which HCl(693 μL, 1M, 10.5 eq.) was added to quench the reaction. The organiclayer was extracted with DCM (3×10 mL). Organic layers combined, dried(Na₂SO₄), and concentrated. The hydrolyzed residue was carried to thenext step without further purification. A vial was charged withhydrolyzed S6 (40 mg, 0.06 mmol), TSTU (54 mg, 0.180 mmol, 3 eq.), DIEA(105 μL, 0.60 mmol, 10 eq.) and DMF (500 μL). Contents were stirred atroom temperature and ambient atmosphere for 24 h, after which thesolvent was concentrated to dryness, and the crude product was purifiedby silica gel chromatography (0-70% Hexanes/EtOAc, linear gradient) toafford PC-JF549-NHS (10 mg, 22%). ¹H NMR (CD₃CN, 400 MHz) δ8.25 (dd,J=8.0, 1.5 Hz, 1H), 8.13 (dd, J=8.0, 0.8 Hz, 1H), 7.73 (dd, J=1.5, 0.7Hz, 1H), 7.54 (dq, J=8.9, 2.0 Hz, 1H), 7.22 (dd, J=8.9, 2.2 Hz, 1H),7.09 (d, J=2.1 Hz, 1H), 6.75 (d, J=1.0 Hz, 1H), 6.69 (d, J=8.4 Hz, 2H),6.12 (q, J=2.3 Hz, 3H), 6.09 (d, J=2.3 Hz, 1H), 3.85 (t, J=7.3 Hz, 8H),2.81 (s, 4H), 2.32 (p, J=7.3 Hz, 4H).

8-(3′,6′-di(azetidin-1-yl)-3-oxo-2-(2-oxo-4-(trifluoromethyl)-2H-chromen-7-yl)spiro[isoindoline-1,9′-xanthene]-6-carboxamido)octanoicacid (S8): A vial was charged with PC-JF549-NHS (5 mg, 0.007 mmol),8-aminooctanoic acid (3.30 mg, 0.021 mmol, 3 eq.), DIEA (11 μL, 0.066mmol, 10 eq.) and DMF (300 μL). Contents were stirred at roomtemperature and ambient atmosphere for 24 h, after which the solvent wasconcentrated to dryness, and the crude product was purified by reversephase HPLC (10-95% MeCN/H₂O, linear gradient, with constant 0.1% v/v TFAadditive; 23 min run, 42 mL/min flow, detection at 550 nm) to afford S8(5 mg, 94%). ¹H NMR (CD₃CN, 400 MHz) δ8.05 (dd, J=7.9, 0.7 Hz, 1H), 7.89(dd, J=7.9, 1.5 Hz, 1H), 7.49 (dt, J=8.9, 2.0 Hz, 1H), 7.36 (d, J=1.2Hz, 1H), 7.29 (dd, J=8.9, 2.2 Hz, 1H), 7.15 (dd, J=9.7, 2.1 Hz, 1H),6.66 (s, 1H), 6.57 (dd, J=8.6, 2.4 Hz, 2H), 6.20-6.10 (m, 3H), 6.04 (m,2H), 3.90 (t, J=7.3 Hz, 8H), 3.36 (q, J=6.7 Hz, 2H), 2.35 (dq, J=22.4,7.3 Hz, 6H), 1.57 (dt, J=20.0, 6.9 Hz, 4H), 1.31 (s, 6H).

(2aR,4S,4aS,6R,9S,11S,12S,12bS)-12b-acetoxy-9-(((2R,3S)-3-(8-(3′,6′-di(azetidin-1-yl)-3-oxo-2-(2-oxo-4-(trifluoromethyl)-2H-chromen-7-yl)spiro[isoindoline-1,9′-xanthene]-6-carboxamido)octanamido)-2-hydroxy-3-phenylpropanoyl)oxy)-4,6,11-trihydroxy-8,13,13-trimethyl-5-oxo-2a,3,4,4a,5,6,9,10,11,12,12a,12b-dodecahydro-1H-[7,11]methanocyclodeca[3,4]benzo[1,2-b]oxet-12-ylbenzoate (PC-JF549-Tx): A vial was charged with S8 (4 mg, 0.005 mmol),Tx⁵ (9.2 mg, 0.012 mmol, 2.5 eq.), HATU (3.7 mg, 0.01 mmol, 2 eq.), DIEA(43 μL, 0.250 mmol, 50 eq.) and DMF (500 μL). Contents were stirred atroom temperature and ambient atmosphere for 48 h, after which thesolvent was concentrated to dryness, and the crude product was purifiedby silica gel chromatography (0-10% MeOH/DCM, linear gradient) to affordPC-JF549-Tx (1.5 mg, 20%). LRMS (ESI) calcd for C₈₂H₈₂F₃N₅O₁₈ [M]⁺1496.6, found 1496.5

3. Synthesis of the Photochromic JaneliaFluor Dyes part II (Scheme 2,FIGS. 23-25)

In the previous section, the coumarin switch and the biological handlewere introduced to the fluorophore's molecular structure separately, andat different synthetic stages. This pathway increases the syntheticsteps that include the precious fluorophore increasing the cost of thesynthesis, and limits its generalizability. In another syntheticapproach, the coumarin is designed to incorporate the biological handlein its molecular structure prior to its conjugation to the fluorophore.This reduces the number of synthetic steps with the fluorophoreincluded, potentially reducing the overall synthetic cost, and allowingfor the creation of libraries of fluorophores via conjugating thisuniversal switch to existing fluorophores.

To prepare the universal coumarin switch, S5, the commercially acquired4-bromo-2-hydroxybenzaldehyde was condensed with tent-butyl ethylmalonate to give the coumarin S1.⁶ Palladium catalyzed cross-couplingwith benzyl carbamate provided S2, whose carboxyl functionality, at the3′-position, can be selectively de-protected with TFA to get S3.Activation of S3 with DCC and then amidation with a HTL, or any otherbiological handle, yielded S4, which upon reduction with hydrogen gas inthe presence of a palladium catalyst produced the universal switch S5.The amine on S5 can now be condensed with any fluorophore of interest,for example JF549, scheme 2 inset, resulting in PC(II)-JF549-HY which isnow rendered both photochromic and capable of labeling a specificprotein-of-interest.

Unlike AlexaFluor dyes which are highly polar, JaneliaFluor dyes do notcarry a net charge, and hence are cell-permeant and compatible withlive-cell imaging. However, this comes with lower water-solubility. Byadding an additional hydrophobic switch—the coumarin, water solubilitycan be further reduced, with potential detrimental effects on cellularlabeling specificity as it introducing higher affinity to hydrophobicpockets. In scheme 2, we also demonstrate a pathway to introduce watersolubilizing groups into the coumarin structure, prior to itsconjugation to the JaneliaFluor, further demonstrating its versatility.

Activation of the carboxylic acid at the 3′-position of S3 with EDC inthe presence of HOBT, and then amidation with PEG-NH₂ (Scheme 2 inset)produced intermediate S6, which now features the solubilizing PEG group,and the protected amine. It is also possible to introduce other types ofsolubilizing groups using the same approach. Removal of the carbamateprotecting group in S6, yields S7, which can now be condensed to aJaneliaFluor dye, JF646-6-OMe to produce S8 which can now be hydrolyzedunder basic conditions and then coupled to a biological handle such as aHTL as in PC-JF646-PEG-HT, or an NHS ester PC-JF646-PEG-NHS. The latterNHS activated JaneliaFluor dye is more suitable for immunostaining infixed cells, due to its better water solubility, compared toPC-JF549-NHS, because of its enhanced water solubility.

4. General Experimental Information for Synthesis of Compounds forScheme 2

tert-butyl 7-bromo-2-oxo-2H-chromene-3-carboxylate (S1): A roundbottomed flask was charged with 4-bromo-2-hydroxybenzaldehyde (2.0 g,1.00 mmol), tert-butyl ethyl malonate (2.06 g, 1.10 mmol, 1.1 eq.),piperidine (0.15 mL, 0.15 mmol, 0.15 eq.) and acetonitrile (3.00 mL).The contents were stirred at room temperature and argon atmosphere for24 hours, after which the solvent was concentrated to dryness, and thecrude product purified via silica gel chromatography (1:1 Hexanes/DCM,linear gradient) to afford S1 (2.03 g, 63%). ¹H NMR (CDCl₃, 400 MHz)δ8.34 (d, J=0.7 Hz, 1 H), 7.53 (dt, J=1.6, 0.8 Hz, 1H), 7.48-7.40 (m,2H), 1.62 (s, 9H).

tert-butyl7-(((benzyloxy)carbonyl)amino)-2-oxo-2H-chromene-3-carboxylate (S2): Avial was charged with S1 (1.0 g, 3.08 mmol), benzyl carbamate (0.93 g,6.15 mmol, 2 eq.), Pd₂dba₃ (0.14 g, 0.15 mmol, 0.05 eq.), CsCO₃ (1.5 g,4.61 mmol, 1.5 eq.) Xantphos (0.27 g, 0.46 mmol, 0.15 eq.). The vial wassealed and evacuated/backfilled with argon (3×). Toluene (6.0 mL) wasadded, and the reaction was flushed again with argon (3×). The reactionwas then stirred at 100° C. for 24 h. It was then cooled down to RT,filtered through celite with DCM, concentrated to dryness, and the crudeproduct purified via silica gel chromatography (0-50% EtOAc/Hexanes,linear gradient) to afford S2 (0.48 g, 39%). ¹H NMR (CDCl₃, 400 MHz):δ8.34 (s, 1H), 7.50 (d, J=8.6 Hz, 1H), 7.47 (d, J=2.0 Hz, 1H), 7.43-7.33(m, 6H), 6.96 (s, 1H), 5.23 (d, J=1.1 Hz, 2H), 1.59 (s, 9H).

7-(((benzyloxy)carbonyl)amino)-2-oxo-2H-chromene-3-carboxylic acid (S3):A round bottomed flask was charged with S2 (1.74 g, 4.40 mmol), TFA(1.35 mL, 17.6 mmol, 4 eq.) and DCM (45 mL). The contents were stirredat room temperature and ambient atmosphere for 24 hours, after whichtoluene (10 mL) was added, and the DCM evaporated under reducedpressure. MeOH (10×2 mL) was then added to azeotropically remove thetoluene. The crude product S3 was then used in the next step withoutfurther purification. ¹H NMR (MeOD-d₄, 400 MHz): δ10.13 (s, 1H), 8.77(s, 1H), 7.88 (s, 1H), 7.79-7.67 (m, 2H), 7.50-7.23 (m, 6H), 5.23 (s,2H).

benzyl(3-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-oxo-2H-chromen-7-yl)carbamate(S4): A vial was charged with S3 (0.7 g, 2.06 mmol), HTL (0.55 g, 2.48mmol, 1.2 eq.), EDC (0.47 g, 2.48 mmol, 1.2 eq.), HOBT (0.33 g, 2.48mmol, 1.2 eq.) and DMF (9 mL). The reaction was then stirred at roomtemperature and ambient atmosphere for 24 h. The organic layer wasextracted with DCM (3×30 mL). Organic layers combined, dried (Na₂SO₄),and concentrated. The crude product was purified via silica gelchromatography (0-30% EtOAc/DCM, linear gradient) to afford S4 (1.09 g,96%). ¹H NMR (CDCl₃, 400 MHz): δ8.98 (s, 1H), 8.74 (s, 1H), 7.67 (s,1H), 7.61-7.49 (m, 2H), 7.43-7.27 (m, 6H), 5.22 (s, 2H), 3.67-3.55 (m,8H), 3.32-3.40 (m, 4H), 1.80-1.67 (m, 2H), 1.63-1.53 (m, 2H), 1.47-1.29(m, 4H).

7-amino-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-oxo-2H-chromene-3-carboxamide(S5): A round bottomed flask was charged with S4 (1.10 g, 2.02 mmol),Pd/C (60 mg, 0.61 mmol, 0.3 eq.) and EtOAc (3 mL). The flask was sealedwith a rubber septum and evacuated/backfilled first with argon (3×),then with hydrogen gas (3×) via a balloon. The reaction was then stirredat room temperature and hydrogen atmosphere (via a balloon) for 48hours. The reaction mixture was filtered over celite, washed with EtOAc(3×10 mL), concentrated, and the crude product was purified via silicagel chromatography (0-50% EtOAc/DCM, linear gradient) to afford S5 (0.26g, 32%). ¹H NMR (CDCl₃, 400 MHz): δ8.97 (s, 1H), 8.69 (s, 1H), 7.39 (d,J=8.0 Hz, 1H), 6.60 (dt, J=8.2, 4.2 Hz, 1H), 6.55 (s, 1H), 4.53 (s, 2H),3.67-3.57 (m, 8H), 3.54-3.44 (m, 4H), 1.75 (p J=7.1 Hz, 2H), 1.62-1.50(m, 2H), 1.47-1.30 (m, 4H).

N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-7-(3′,6′-di(azetidin-1-yl)-3-oxospiro[isoindoline-1,9′-xanthen]-2-yl)-2-oxo-2H-chromene-3-carboxamide(PC(II)-JF549-HT): A vial was charged with JF549¹ (25 mg, 0.061 mmol),S5 (75 mg, 0.183 mmol, 3 eq.), HATU (70 mg, 0.183 mmol, 3 eq.), DIEA(106 μL, 0.61 mmol, 10 eq.) and DMF (300 μL). Contents were stirred atroom temperature and ambient atmosphere for 24 h, after which thesolvent was concentrated to dryness, and the crude product was purifiedvia reverse phase HPLC (10-95% MeCN/H₂O, linear gradient, with constant0.1% v/v TFA additive; 23 min run, 42 mL/min flow, detection at 550 nm)to afford PC(II)-JF549-HT (3 mg, 6%). ¹H NMR (CDCl₃, 400 MHz): δ8.89 (s,1H), 8.74 (s, 1 H), 8.00 (dd, J=6.8, 1.9 Hz, 1H), 7.60-7.52 (m, 2H),7.50 (d, J=8.6 Hz, 1H), 7.35 (dd, J=8.6, 2.0 Hz, 1H), 7.29 (d, J=2.0 Hz,1H), 7.07 (dd, J=6.5, 1.8 Hz, 1H), 6.64 (d, J=8.5 Hz, 2H), 6.17 (d,J=2.3 Hz, 2H), 6.07 (dd, J=8.6, 2.3 Hz, 2H), 3.90 (t, J=7.2 Hz, 8H),3.80-3.69 (m, 2H), 3.69-3.51 (m, 4H), 3.57 (t, J=6.8 Hz, 2H), 3.48 (t,J=6.7 Hz, 2H), 3.20 (qd, J=7.4, 3.4 Hz, 2H), 2.38 (p, J=7.2 Hz, 4H),1.84-1.76 (m, 2H), 1.76-1.56 (m, 2H), 1.42-1.36 (m, 4H).

benzyl(3-((2,5,8,11-tetraoxatridecan-13-yl)carbamoyl)-2-oxo-2H-chromen-7-yl)carbamate(S6): A vial was charged with S3 (50 mg, 0.15 mmol), PEG-NH₂ (37 mg,0.18 mmol, 1.2 eq.), EDC (34 mg, 0.18 mmol, 1.2 eq.), HOBT (24 mg, 0.18mmol, 1.2 eq.) and DMF (3 mL). The reaction was then stirred at roomtemperature and ambient atmosphere for 24 h. The reaction wasconcentrated. The crude product was purified via silica gelchromatography (0-10% MeOH/DCM, linear gradient) to afford S6 (67 mg,85%). ¹H NMR (CDCl₃, 400 MHz): δ9.01 (s, 1H), 8.72 (s, 1H), 8.21 (s,1H), 7.73 (s, 1H), 7.50 (d, J=8.6 Hz, 1H), 7.40-7.30 (m, 6H), 5.22 (s,2H), 3.70-3.57 (m, 14H), 3.52 (t, J=4.5 Hz, 2H), 3.34 (s, 3H).

7-amino-2-oxo-N-(2,5,8,11-tetraoxatridecan-13-yl)-2H-chromene-3-carboxamide(S7): A round bottomed flask was charged with S6 (67 mg, 0.13 mmol),Pd/C (2 mg, 0.13 mmol, 0.1 eq.) and EtOAc (3 mL). The flask was sealedwith a rubber septum and and evacuated/backfilled first with argon (3×),then with hydrogen gas (3×) via a balloon. The reaction was then stirredat room temperature and hydrogen atmosphere (via a balloon) for 48hours. The reaction mixture was deposited on celite, concentrated, andthe crude product was purified via silica gel chromatography (0-10%MeOH/DCM, linear gradient) to afford S7 (33 mg, 65%). ¹H NMR (CDCl₃, 400MHz): δ8.97 (t, J=5.3 Hz, 1H), 8.61 (s, 1H), 7.33 (d, J=8.5 Hz, 1H),6.59 (dd, J=8.5, 2.2 Hz, 1H), 6.54 (d, J=2.1 Hz, 1H), 4.96 (s, 2H),3.75-3.58 (m, 14H), 3.58-3.48 (m, 2H), 3.38 (s, 3H).

methyl2′-(3-((2,5,8,11-tetraoxatridecan-13-yl)carbamoyl)-2-oxo-2H-chromen-7-yl)-3,7-di(azetidin-1-yl)-5,5-dimethyl-3′-oxo-5H-spiro[dibenzo[b,e]siline-10,1′-isoindoline]-6′-carboxylate(S8): A vial was charged with JF646-6-OMe (12 mg, 0.023 mmol), oxalylchloride (3 μL, 0.028 mmol, 1.2 eq.) and DCM (2.00 mL) and the contentwas stirred at room temperature and ambient atmosphere for 30 minutes.S7 (28 mg, 0.070 mmol, 3 eq.) and DIEA (42 μL, 0.235 mmol, 10 eq.) werethen added and the contents were stirred for 1 h, after which thesolvent was concentrated to dryness, and the crude product was purifiedby silica gel chromatography (0-5% MeOH/DCM, linear gradient) to affordS7 (7.2 mg, 35%). ¹H NMR (CDCl₃, 400 MHz): δ8.98 (d, J=6.0 Hz, 1H), 8.68(d, J=0.7 Hz, 1H), 8.01 (d, J=1.0 Hz, 2H), 7.68 (dd, J=8.7, 2.1 Hz, 1H),7.64 (d, J=2.1 Hz, 1H), 7.55-7.49 (m, 1H), 7.38 (d, J=8.8 Hz, 1H), 6.76(d, J=8.8 Hz, 2H), 6.61 (d, J=2.7 Hz, 2H), 6.23 (dd, J=8.8, 2.6 Hz, 2H),3.88 (t, J=7.3 Hz, 8H), 3.81 (s, 3H), 3.70-3.60 (m, 14H), 3.58-3.51 (m,2H), 3.37 (s, 3H), 2.35 (p, J=7.2 Hz, 4H), 0.71 (s, 3H), 0.57 (s, 3H).

2,5-dioxopyrrolidin-1-yl2′-(3-((2,5,8,11-tetraoxatridecan-13-yl)carbamoyl)-2-oxo-2H-chromen-7-yl)-3,7-di(azetidin-1-yl)-5,5-dimethyl-3′-oxo-5H-spiro[dibenzo[b,e]siline-10,1′-isoindoline]-6′-carboxylate(PC-JF646-PEG-NHS): A vial was charged with S8 (7 mg, 0.008 mmol), NaOH(80 μL, 1M, 10 eq.) MeOH (1.0 mL) and THF (0.5 mL). Content was stirredat room temperature and ambient atmosphere for 24 h, after which HCl (85μL, 1M, 10.5 eq.) was added to quench the reaction. The organic layerwas extracted with DCM (3×10 mL). Organic layers combined, dried(Na₂SO₄), and concentrated. The hydrolyzed residue was carried to thenext step without further purification. A vial was charged withhydrolyzed S8 (6.8 mg, 0.008 mmol), TSTU (7 mg, 0.023 mmol, 3 eq.), DIEA(14 μL, 0.08 mmol, 10 eq.) and DMF (300 μL). Contents were stirred atroom temperature and ambient atmosphere for 24 h, after which thesolvent was concentrated to dryness, and the crude product was purifiedby silica gel chromatography (0-70% Acetone/DCM, linear gradient) toafford PC-JF646-PEG-NHS (1.3 mg, 17%). LRMS (ESI) calcd forC₅₈H₇₂ClN₅O₁₁Si [M]⁺ 1078.8, found 1078.5

2′-(3-((2,5,8,11-tetraoxatridecan-13-yl)carbamoyl)-2-oxo-2H-chromen-7-yl)-3,7-di(azetidin-1-yl)-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-5,5-dimethyl-3′-oxo-5H-spiro[dibenzo[b,e]siline-10,1′-isoindoline]-6′-carboxamide(PC-JF646-PEG-HT): A vial was charged with S8 (14 mg, 0.016 mmol), NaOH(160 μL, 1M, 10 eq.) MeOH (1.0 mL) and THF (0.5 mL). Content was stirredat room temperature and ambient atmosphere for 24 h, after which HCl(168 μL, 1M, 10.5 eq.) was added to quench the reaction. The organiclayer was extracted with DCM (3×10 mL). Organic layers combined, dried(Na₂SO₄), and concentrated. The hydrolyzed residue was carried to thenext step without further purification. A vial was charged withhydrolyzed S8 (14 mg, 0.016 mmol), HTL (16 mg, 0.048, 3 eq.), HATU (18mg, 0.048 mmol, 3 eq.), DIEA (28 μL, 0.16 mmol, 10 eq.) and DMF (500μL). Contents were stirred at room temperature and ambient atmospherefor 24 h, after which the solvent was concentrated to dryness, and thecrude product was purified by silica gel chromatography (0-5% MeOH/DCM,linear gradient) to afford PC-JF646-PEG-HT (13 mg, 75%). ¹H NMR (CDCl₃,400 MHz): δ8.98 (d, J=5.6 Hz, 1H), 8.67 (s, 1H), 7.99 (s, 1H), 7.69 (dd,J=8.0, 1.4 Hz, 1H), 7.66-7.61 (m, 2H), 7.37 (d J=9.6 Hz, 1H), 7.29 (s,1H), 6.75 (d, J=8.7 Hz, 2H), 6.64 (t, J=5.4 Hz, 1H), 6.60 (d, J=2.6 Hz,2H), 6.23 (dd, J=8.8, 2.6 Hz, 2H), 3.88 (t, J=7.2 Hz, 8H), 3.76-3.49 (m,26H), 3.40 (t, J=6.6 Hz, 2H), 3.37 (s, 3H), 2.35 (p, J=7.2 Hz, 4H),1.83-1.70 (m, 2H), 1.58-1.48 (m, 2H), 1.37-1.26 (m, 4H), 0.69 (s, 3H),0.55 (s, 3H).

5. Synthesis of the Photochromic AlexaFluor Dyes (Scheme 3, FIGS. 25-26)

To further demonstrate the generalizability of this approach in inducingphotochromism in fluorophores, these coumarin switches were introducedinto the molecular structure of AlexaFluor 594, Scheme 3, at the sameposition with the JaneliaFluor dyes. The chemistry can be extended toother AlexaFluor dyes as well. AlexaFluor dyes are more polar and hencewater soluble, so they are ideal antibody labels for immunofluorescence.

The synthesis of PC-AF549-NHS, starts with the advanced intermediates,S1 and S2, which were synthesized from the condensation of thecommercially available 1,2-Dihydro-1,2,2,4-tetramethyl-7-quinolinol;1,2-Dihydro-1,2,2,4-tetramethylquinolin-7-ol and trimellitic anhydrideas described elsewhere.⁷ The 5′- and 6′-carboxy isomers were separatedby reverse phase HPLC (30-60% MeCN/H₂O, linear gradient, with constant0.1% v/v TFA additive; 23 min run, 42 mL/min flow, detection at 500 nm).The 6′-isomer eluting after 11.75 mins, and the 5′-isomer eluting after12.5 mins. The combined yield of the two isomers was 26%, and the5′-isomer was used in the synthesis of PC-AF594-NHS, while the 6′-isomerwas used in the synthesis of the PC-AF594-HT. S1 and S2 were thenmethylated by first activation with DSC to yield the respective NHSester, followed by trapping with sodium methoxide to yield the methylesters. Sulfonating the methyl esters S3 and S4 in conc. sulfuric acidresulted in the sulfonated rhodamines (AlexaFluor549 derivatives) whichwere then treated with HATU to activate the carboxylate at the3′-position before amidation with 7-Amino-4-(trifluoromethyl) coumarinto yield photochromic isomers S7 and S8. Upon hydrolysis of the methylesters in basic conditions, the hydrolyzed 5′-isomer (S9) was then usedto install an NHS ester functionality producing PC-AF594-NHS forantibody labeling, while the hydrolyzed 6′-isomer (S10) was used toinstall a HTL to produce PC-AF594-HT for labeling HaloTag protein.

Through similar chemical transformations, it is also possible to attachother biological handles to the photochromic AF594, and otherAlexafluors, such as SNAP-Tag or click-chemistry substrates, which willincrease the number of biological targets that can be specificallylabeled with these fluorophores.

6. General Experimental Information for Synthesis of Compounds forScheme 3 (FIGS. 26-27)

5-(methoxycarbonyl)-2-(1,2,2,4,8,10,10,11-octamethyl-10,11-dihydro-2H-pyrano[3,2-g:5,6-g′]diquinolin-1-ium-6-yl)benzoate(S3): A vial was charged with S1 (50 mg, 0.088 mmol), DSC (68 mg, 0.267mmol, 3 eq.), DMAP (1 mg, 0.009 mmol, 0.1 eq.), Et₃N (73 μL, 0.528 mmol,6 eq.) and DMF (300 μL). Contents were stirred at room temperature andambient atmosphere for 1 h, after which NaOMe (77 μL, 0.355 mmol, 25%wt/wt in MeOH, 4 eq.) and the mixture stirred for 24 h. Solvent wasconcentrated to dryness, and the crude product was purified by reversephase HPLC (20-80% MeCN/H₂O, linear gradient, with constant 0.1% v/v TFAadditive; 23 min run, 42 mL/min flow, detection at 050 nm) to afford S3(30 mg, 60%). LRMS (ESI) calcd for C₃₆H₃₆N₂O₅ [M]⁺576.7, found 577.3

4-(methoxycarbonyl)-2-(1,2,2,4,8,10,10,11-octamethyl-10,11-dihydro-2H-pyrano[3,2-g:5,6-g′]diquinolin-1-ium-6-yl)benzoate(S4): A vial was charged with S2 (30 mg, 0.053 mmol), DSC (41 mg, 0.156mmol, 3 eq.), DMAP (0.7 mg, 0.005 mmol, 0.1 eq.), Et₃N (44 μL, 0.32mmol, 6 eq.) and DMF (300 μL). Contents were stirred at room temperatureand ambient atmosphere for 1 h, after which NaOMe (48 μL, 0.213 mmol,25% wt/wt in MeOH, 4 eq.) and the mixture stirred for 24 h. Solvent wasconcentrated to dryness, and the crude product was purified by reversephase HPLC (20-80% MeCN/H₂O, linear gradient, with constant 0.1% v/v TFAadditive; 23 min run, 42 mL/min flow, detection at 050 nm) to afford S4(19 mg, 60%). LRMS (ESI) calcd for C₃₆H₃₆N₂O₅[M]⁺ 576.7, found 577.2.

2-(1,2,2,10,10,11-hexamethyl-4,8-bis(sulfomethyl)-10,11-dihydro-2H-pyrano[3,2-g:5,6-g′]diquinolin-1-ium-6-yl)-5-(methoxycarbonyl)benzoate(S5): A vial was charged with S3 (30 mg, 0.052 mmol) and conc. H₂SO₄ (2mL). Contents were stirred at room temperature and ambient atmospherefor 72 h, after which the reaction was cooled down to 0° C. and coolwater was added dropwise to dilute the mixture before loading it on areverse phase C18 silica gel chromatography column. The column waswashed with copious amounts of water until the pH of the filtrate isadjusted to around 5. The sulfonated rhodamine was then eluted withCH₃CN to afford S5 (20 mg, 38%). LRMS (ESI) calcd for C₃₆H₃₆N₂O₁₁S₂ [M]⁺736.8, found 737.3

2-(1,2,2,10,10,11-hexamethyl-4,8-bis(sulfomethyl)-10,11-dihydro-2H-pyrano[3,2-g:5,6-g′]diquinolin-1-ium-6-yl)-4-(methoxycarbonyl)benzoate(S6): A vial was charged with S4 (18 mg, 0.031 mmol) and conc. H₂SO₄ (1mL). Contents were stirred at room temperature and ambient atmospherefor 72 h, after which the reaction was cooled down to 0° C. and coolwater was added dropwise to dilute the mixture before loading it on areverse phase C18 silica gel chromatography column. The column waswashed with copious amounts of water until the pH of the filtrate isadjusted to around 5. The sulfonated rhodamine was then eluted withCH₃CN to afford S6 (22 mg, 96%). LRMS (ESI) calcd for C₃₆H₃₆N₂O₁₁S₂ [M]⁺736.8, found 737.3

(5-(methoxycarbonyl)-1′,2′,2′,10′,10′,11′-hexamethyl-3-oxo-2-(2-oxo-4-(trifluoromethyl)-2H-chromen-7-yl)-1′,2′,10′,11′-tetrahydrospiro[isoindoline-1,6′-pyrano[3,2-g:5,6-g′]diquinoline]-4′,8′-diyl)dimethanesulfonicacid (S7): A vial was charged with S5 (20 mg, 0.027 mmol),7-amino-4-(trifluoromethyl)coumarin (19 mg, 0.081 mmol, 3 eq.), HATU (31mg, 0.081 mmol, 3 eq.), Et₃N (76 μL, 0.543 mmol, 20 eq.) and DMF (500μL).

Contents were stirred at room temperature and ambient atmosphere for 24h, after which the solvent was concentrated to dryness, and the crudeproduct was purified via reverse phase HPLC (10-95% MeCN/H₂O, lineargradient, with constant 0.1% v/v TFA additive; 23 min run, 42 mL/minflow, detection at 550 nm) to afford S7 (11 mg, 42%). LRMS (ESI) calcdfor C₄₆H₄₀F₃N₃O₁₂S₂ [M]⁺ 948.0, found 948.1

(6-(methoxycarbonyl)-1′,2′,2′,10′,10′,11′-hexamethyl-3-oxo-2-(2-oxo-4-(trifluoromethyl)-2H-chromen-7-yl)-1′,2′,10′,11′-tetrahydrospiro[isoindoline-1,6′-pyrano[3,2-g:5,6-g′]diquinoline]-4′,8′-diyl)dimethanesulfonic acid (S8): A vial was charged with S6 (20 mg,0.027 mmol), 7-amino-4-(trifluoromethyl)coumarin (19 mg, 0.081 mmol, 3eq.), HATU (31 mg, 0.081 mmol, 3 eq.), Et₃N (76 μL, 0.543 mmol, 20 eq.)and DMF (500 μL). Contents were stirred at room temperature and ambientatmosphere for 24 h, after which the solvent was concentrated todryness, and the crude product was purified via reverse phase HPLC(10-95% MeCN/H₂O, linear gradient, with constant 0.1% v/v TFA additive;23 min run, 42 mL/min flow, detection at 550 nm) to afford S7 (8 mg,31%). LRMS (ESI) calcd for C₄₆H₄₀F₃N₃O₁₂S₂ [M]⁺ 948.0, found 948.1

1′,2′,2′,10′,10′,11′-hexamethyl-3-oxo-2-(2-oxo-4-(trifluoromethyl)-2H-chromea-7-yl)-4′,8′-bis(sulfomethyl)-1′,2′,10′,11′-tetrahydrospiro[isoindoline-1,6′-pyrano[3,2-g:5,6-g′]diquinoline]-5-carboxylicacid (S9): A vial was charged with S7 (11 mg, 0.011 mmol), NaOH (110 μL,1M, 10 eq.) and MeOH (1.0 mL). Content was stirred at room temperatureand ambient atmosphere for 24 h, after which HCl (116 μL, 1M, 10.5 eq.)was added to quench the reaction and the content stirred for 2 h. Thecontent was concentrated and the hydrolyzed residue was purified byreverse phase HPLC (30-70% MeCN/H₂O, linear gradient, with constant 0.1%v/v TFA additive; 23 min run, 42 mL/min flow, detection at 550 nm) toafford S9 (5.5 mg, 51%). LRMS (ESI) calcd for C₄₅H₃₈F₃N₃O₁₂S₂ [M]⁺933.9, found 934.1

1′,2′,2′,10′,10′,11′-hexamethyl-3-oxo-2-(2-oxo-4-(trifluoromethyl)-2H-chromen-7-yl)-4′,8′-bis(sulfomethyl)-1′,2′,10′,11′-tetrahydrospiro[isoindoline-1,6′-pyrano[3,2-g:5,6-g′]diquinoline]-6-carboxylicacid (S10): A vial was charged with S8 (8 mg, 0.008 mmol), NaOH (80 μL,1M, 10 eq.) and MeOH (1.0 mL). Content was stirred at room temperatureand ambient atmosphere for 24 h, after which HCl (88 μL, 1M, 10.5 eq.)was added to quench the reaction and the content stirred for 2 h. Thecontent was concentrated and the hydrolyzed residue was purified byreverse phase HPLC (10-90% MeCN/H₂O, linear gradient, with constant 0.1%v/v TFA additive; 23 min run, 42 mL/min flow, detection at 550 nm) toafford S9 (2 mg, 25%). LRMS (ESI) calcd for C₄₅H₃₈F₃N₃O₁₂S₂ [M]⁺ 933.9,found 934.3

(5-(((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)-1′,2′,2′,10′,10′,11′-hexamethyl-3-oxo-2-(2-oxo-4-(trifluoromethyl)-2H-chromen-7-yl)-1′,2′,10′,11′-tetrahydrospiro[isoindoline-1,6′-pyrano[3,2-g:5,6-g′]diquinoline]-4′,8′-diyl)dimethanesulfonicacid (PC-AF594-NHS): A vial was charged with S9 (1 mg, 0.001 mmol), TSTU(0.5 mg, 0.0015 mmol, 1.5 eq.), DIEA (0.6 μL, 0.003 mmol, 3 eq.) and DMF(50 μL). Contents were stirred at room temperature and ambientatmosphere for 1 h, after which the solvent was diluted with CH₃CN todryness, and the purified by reverse phase HPLC (10-90% MeCN/H₂O, lineargradient, in 10 mM triethylammonium formate buffer, 23 min run, 42mL/min flow, detection at 550 nm) to afford afford PC-AF594-NHS (1 mg,99%). LRMS (ESI) calcd for C₄₉H₄₁F₃N₄O₁₄S2 [M]⁺ 1031.0, found 1031.2

(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-1′,2′,2′,10′,10′,11′-hexamethyl-3-oxo-2-(2-oxo-4-(trifluoromethyl)-2H-chromen-7-yl)-1′,2′,10′,11′-tetrahydrospiro[isoindoline-1,6′-pyrano[3,2-g:5,6-g′]diquinoline]-4′,8′-diyl)dimethanesulfonicacid (PC-AF594-HT): A vial was charged with S10 (2 mg, 0.002 mmol), HTL(3 mg, 0.006, 3 eq.), HATU (3 mg, 0.006 mmol, 3 eq.), DIEA (4 μL, 0.02mmol, 10 eq.) and DMF (300 μL). Contents were stirred at roomtemperature and ambient atmosphere for 24 h, after which the solvent wasconcentrated to dryness, and the crude product was purified by reversephase HPLC (10-90% MeCN/H₂O, linear gradient, with constant 0.1% v/v TFAadditive; 23 min run, 42 mL/min flow, detection at 550 nm) to affordPC-AF594-HT (1.2 mg, 49%). LRMS (ESI) calcd for C₅₅H₅₈ClF₃N₄O₁₃S₂ [M]⁺1139.7, found 1139.3

7. Synthesis of the Photochromic Azepine Dyes (Scheme 4)

Finally, it is also possible to render rhodamine dyes with differentamino substituents on their xanthene core photochromic by introducingthe coumarin switch. Rhodamine with an azepane ring (S1), and rhodamine640 perchlorate (S2), Scheme 4, were activated by HATU, prior toamidation with 7-amino-4-(trifluoromethyl) coumarin in the presence ofHiinig's base.

Scheme 1. Synthesis of PC-Azep. a. HATU, DIEA, DMF,7-amino-4-(trifluoromethyl) coumarin, RT, 24 h.

8. General Experimental Information for Synthesis of Compounds forScheme 4

3′,6′-di(azepan-1-yl)-2-(2-oxo-4-(trifluoromethyl)-2H-chromen-7-yl)spiro[isoindoline-1,9′-xanthen]-3-one(PC-Azep): A vial was charged with S1¹ (25 mg, 0.05 mmol),7-amino-4-(trifluoromethyl) coumarin (23 mg, 0.10 mmol, 3 eq.), HATU (23mg, 0.06 mmol, 1.2 eq.), DIEA (26 μL, 0.151 mmol, 3 eq.) and DMF (1 mL).Contents were stirred at room temperature and ambient atmosphere for 24h, after which the solvent was concentrated to dryness, and the crudeproduct was purified via silica gel chromatography (0-10% MeOH/DCM,linear gradient) to afford PC-Azep (15 mg, 42%). ¹H NMR (CDCl₃, 400 MHz)δ7.90 (d, J=8 Hz, 1H), 7.52-7.38 (m, 3H), 7.32-7.23 (m, 2H), 7.01 (dd,J=6.9, 1.4 Hz, 1H), 6.59-6.49 (m, 3H), 6.33 (d, J=2.6 Hz, 2H), 6.25 (dd,J=8.9, 2.6 Hz, 2H), 5.25 (s, 1H), 3.34 (t, J=6.0 Hz, 8H), 1.74-1.63 (m,8H), 1.60-1.46 (m, 8H).

(PC-Rh₆₄₀): A vial was charged with S2 (50 mg, 0.08 mmol),7-amino-4-(trifluoromethyl) coumarin (30 mg, 0.17 mmol, 2 eq.), HATU (39mg, 0.10 mmol, 1.2 eq.), DIEA (42 μL, 0.24 mmol, 3 eq.) and DMF (1 mL).Contents were stirred at room temperature and ambient atmosphere for 24h, after which the solvent was concentrated to dryness, and the crudeproduct was purified via silica gel chromatography (0-10% MeOH/DCM,linear gradient) to afford PC-Rh₆₄₀ (30 mg, 55%).

REFERENCES

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1. A compound of the following structure:

wherein, X is O, N-alkyl, S, Si(alkyl)₂ or C(alkyl)₂; Y₁ is O, OH, NH₂,N(alkyl)₂ or one of the moieties shown in FIG. 9 (i.e., a, b, c, or d);Y₂ is O, OH, NH₂, N(alkyl)₂ or one of the moieties shown in FIG. 9(i.e., a, b, c, or d); R₁, which can be a substitution at either the 5′position, the 6′ position or both, is hydrogen, C(O)NH-Handle,C(O)-linker-Handle, C(O)NH-Acceptor, C(O)-linker-Acceptor,C(O)NH-linker-CH2-X where X is a leaving group,C(O)NH—(CH₂CH₂)_(n)C(O)N(H)-Handle where “n” is an integer ranging from1 to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” is an integer rangingfrom 1 to 100, C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂-(CH2)₆-Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆—Cl; R₂ ishydrogen, C(O)NH-Handle, C(O)-linker-Handle, C(O)NH-Acceptor,C(O)-linker-Acceptor, C(O)NH-linker-CH2-X where X is a leaving group,C(O)NH—(CH₂CH₂),C(O)N(H)-Handle where “n” is an integer ranging from 1to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” is an integer ranging from1 to 100, C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂-(CH2)₆-Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆—Cl; R₃, R₄,R₅ and R₆ are independently hydrogen, alkyl, —SO₃H, halogen, or R₃ andY₁ can form a ring, or R₄ and Y₁ can form a ring, or R₅ and Y₂ can forma ring, or R₆ and Y₂ can form a ring; A₁ is hydrogen, alkyl, alkenyl,alkynyl, heteroalkyl, heteroalkenyl, cycloalkyl, cycloalkenyl,heterocycloalkyl, heterocycloalkenyl, aromatic, heteroaromatic,substituted alkyl, substituted alkenyl, substituted alkynyl, substitutedheteroalkyl, substituted heteroalkenyl, heterocycloalkyl,heterocycloalkenyl, substituted aromatic group, or substitutedheteroaromatic group; A₂ is hydrogen, alkyl, alkenyl, alkynyl,heteroalkyl, heteroalkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl,heterocycloalkenyl, aromatic, heteroaromatic, substituted alkyl,substituted alkenyl, substituted alkynyl, substituted heteroalkyl,substituted heteroalkenyl, heterocycloalkyl, heterocycloalkenyl,substituted aromatic group, or substituted heteroaromatic group.
 2. Thecompound according to claim 1, wherein X is O, Y₁ is one of the moietiesshown in FIG. 9 (i.e., a, b, c, or d), and Y₂ is one of the moietiesshown in FIG. 9 (i.e., a, b, c, or d).
 3. The compound according toclaim 2, wherein R₂ is a Handle, A₁ is hydrogen, alkyl, or substitutedalkyl.
 4. The compound according to claim 3, wherein A₂ is is hydrogen,alkyl, or substituted alkyl.
 5. The compound according to claim 4,wherein R₄ and R₅ are SO₃H.
 6. A method of imaging one or more cellularstructures within one or more cells, wherein the method comprises thesteps of: a) labeling one or more cells with a compound of the followingstructure

wherein, X is O, N-alkyl, S, Si(alkyl)₂ or C(alkyl)₂; Y₁ is O, OH, NH₂,N(alkyl)₂ or one of the moieties shown in FIG. 9 (i.e., a, b, c, or d);Y₂ is O, OH, NH₂, N(alkyl)₂ or one of the moieties shown in FIG. 9(i.e., a, b, c, or d); R₁, which can be a substitution at either the 5′position, the 6′ position or both, is hydrogen, C(O)NH-Handle,C(O)-linker-Handle, C(O)NH-Acceptor, C(O)-linker-Acceptor,C(O)NH-linker-CH2-X where X is a leaving group,C(O)NH—(CH₂CH₂)_(n)C(O)N(H)-Handle where “n” is an integer ranging from1 to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” is an integer rangingfrom 1 to 100, C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂-(CH2)₆-Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆—Cl; R₂ ishydrogen, C(O)NH-Handle, C(O)-linker-Handle, C(O)NH-Acceptor,C(O)-linker-Acceptor, C(O)NH-linker-CH2-X where X is a leaving group,C(O)NH—(CH₂CH₂)_(n)C(O)N(H)-Handle where “n” is an integer ranging from1 to 100, C(O)NH—(CHCH)_(n)-Acceptor where “n” is an integer rangingfrom 1 to 100, C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂-(CH2)₆-Cl, orC(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₃—C(O)NH—(CH₂CH₂O)₂—(CH₂)₆—Cl; R₃, R₄,R₅ and R₆ are independently hydrogen, alkyl, —SO₃H, halogen, or R₃ andY₁ can form a ring, or R₄ and Y₁ can form a ring, or R₅ and Y₂ can forma ring, or R₆ and Y₂ can form a ring; A₁ is hydrogen, alkyl, alkenyl,alkynyl, heteroalkyl, heteroalkenyl, cycloalkyl, cycloalkenyl,heterocycloalkyl, heterocycloalkenyl, aromatic, heteroaromatic,substituted alkyl, substituted alkenyl, substituted alkynyl, substitutedheteroalkyl, substituted heteroalkenyl, heterocycloalkyl,heterocycloalkenyl, substituted aromatic group, or substitutedheteroaromatic group; A₂ is hydrogen, alkyl, alkenyl, alkynyl,heteroalkyl, heteroalkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl,heterocycloalkenyl, aromatic, heteroaromatic, substituted alkyl,substituted alkenyl, substituted alkynyl, substituted heteroalkyl,substituted heteroalkenyl, heterocycloalkyl, heterocycloalkenyl,substituted aromatic group, or substituted heteroaromatic group. toprovide one or more labeled cells b) directing at least one beam oflight to the one or more labeled cells, such that a detectable signal isproduced from the one or more labeled cells; c) recording the detectablesignal, thereby imaging one or more structures within the one or morecells.
 7. The method according to claim 6, wherein X of the structure isO, Y₁ is one of the moieties shown in FIG. 9 (i.e., a, b, c, or d), andY₂ is one of the moieties shown in FIG. 9 (i.e., a, b, c, or d).
 8. Themethod according to claim 7, wherein R₂ of the structure is a Handle, A₁is hydrogen, alkyl, or substituted alkyl.
 9. The method according toclaim 8, wherein A₂ is hydrogen, alkyl, or substituted alkyl.
 10. Themethod according to claim 9, wherein R₄ and R₅ are SO₃H.